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Studies on the regulation of p53 and investigation of functional significance of interactions between p53 and its binding proteins

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Studies on the regulation of p53 and investigation of functional significance of interactions between p53 and its binding proteins

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MICROFILMED 2002
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STUDIES ON THE REGULATION OF p53 AND
INVESTIGATION OF FUNCTIONAL SIGNIFICANCE OF
INTERACTIONS BETWEEN p53 AND ITS BINDING PROTEINS
By
KAI-JIN WU
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Molecular Microbiology and Immunology)
December 1999
Copyright 1999 Kai-Jin Wu
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UMI Number: 3054913
Copyright 1999 by
Wu, Kai-Jin
All rights reserved.
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 90007
Vris dissertation, written by
j < / \ \ - J i N JaI u
under the direction of h.&.c. Dissertation
Committee, and approved by all its members,
has been presented to and accepted by The
Graduate School, in partial fulfillment of re
quirements for the degree of
DOCTOR OF PHILOSOPHY
Dean of Graduate Studies
DISSERTATION* COMMITTEE
Date ....November, „ 30?. .19 9 9
I COMMITTEE
y 7 Chairperson
....
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Acknowledgements
I wish to thank my mentor and the chairman of my annual research approval and
thesis committee Dr. Yuen Kai Teddy Fung for his support and guidance. This project
would have not been possible without him. I particularly thank Dr. Barbara Driscoll, and
Dr. Anne T'ang for their important guidance in both science and English, and their
collaborative efforts. I also thank Dr. Duan Yang, graduate student Ling Li, and Dr. Chen
Li for their participation in the project.
I wish to thank all my annual research approval committee and thesis committee
members Dr. Jing-Hsiung James Ou, Dr. Ambrose Yaw-Shong Jong, Dr. Robert Maxson,
and Dr. John Natis for their guidance throughout the years.
I wish to thank my family members Mr. Philbert K. Davalos, Mr. Zi-Yun Wang, Kai-
Yu Wu and Kai-Ling Wu; and professor Mei-Hao Hu, my mentor for my Ph D program
in China, and friends for their support and encouragement.
I am very grateful to my coworkers who provided materials and other supports: Dr.
Kai Wu for providing cell lines and technical information, Dr. Yi-Hui Hu for expression
vector MTneo, Dr. Takashi Koyama for pGEX2Tp53, graduate student Hong-Jun Zhang
for anti-RB monoclonal antibody P I6, Dr. Xiang-He Shi for the cDNA of RBP2, Dr. Fu-
Hui Zhang, Dr. Qing Ji, Dr. Chunli Yan, and Dr. Shumin Wen for their technical
assistance, and Dr. Yuan-Ping Han and Dr. Emmanuel Paul for their discussion and
communication.
I am very grateful to the following professors who generously provided experimental
materials: Dr. Jing-Shiung James Ou for reporter POSTCAT2, Dr. Chi Van Dong for the
ii
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chimeric gene expression vector pGALO and reporter pGAL4 5/E1BCAT, Dr. David
Morgan for cyclin A and Cdk2 baculovirus, Dr. Charles Sherr for cyclin D baculoviruses,
and Dr. John Minna for the tumor cell line H358.
I thank Dr. Ambrose Yaw-Shong Jong and his employees Dr. Luo Feng, graduate
student Ke-Fei Yu, Dr. Bing Wang, and Dr. Yu Hu for their invaluable support during the
time I was writing the thesis.
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iii
TABLE OF CONTENTS
Acknowledgements ii-iii
List of Figures and Tables vi-viii
Abstract ix-x
Chapter 1
Background and significance 1-15
Chapter 2
The carboxy 1-terminus of p53 regulates its DNA
binding and kinase reaction 16-55
Summary 16-18
Introduction 18-20
Results 21-40
Discussion 40-44
Conclusion 44
Materials and Methods 45-55
Chapter 3
Discovery of binding of D-type cyclins to p53 and
Functional study of the interaction 56-82
Summary 56-57
Introduction 57-63
Results 63-75
Discussion 76-78
Conclusion 78-79
Materials and Methods 79-82
Chapter 4
Investigation of functional significance of p53-RBP2
interaction and exploration of RBP2 function 83-120
Summary 83-85
Introduction 85-90
Results 90-108
Discussion 109-113
Conclusion 113
Materials and Methods 114-120
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Chapter 5
Discussion
References
121-122
123-145
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LIST OF FIGURES AND TABLES
FIGURES
Chapter 1
Fig. 1. Homologous sequences P5 and P3 between RB and p53........................14
Chapter 2
Fig. 1. Homologous sequences P5 and P3 between RB and p53....................... 20
Fig. 2. Deletion of P5 (381-393) or mutation of the conserved
amino acid residues within P5 between RB and p53 prohibits
phosphorylation of p53 by Cdk2.......................................................................... 22-23
Fig. 3. Further deletion of p53 from residue 380 recovers
the availability to be phosphorylated on Ser3is by Cdk2.....................................24
Fig. 4. Binding by anti-p53 C-terminal antibodies Pab421 and
Pabl22 doesn’t interfere with phosphorylation by Cdk2....................................26
Fig. 5. Deletion of P5 (381-393) or mutation of the conserved amino
acid residues between RB and p53 within P5 prohibits p53 from
binding the consensusDNA.................................................................................. 27-28
Fig. 6. Immunoprecipitation of wild-type p53 and the mutants to
examine whether mutation or deletion of P5 impairs the recognition
of the protein by anti-P3 antibody Pab421.......................................................... 30-31
Fig. 7. EMSA for testing the effects of mutation of P3 (372-380) and
P2 (357-370) on the sequence-specific DNA-binding activity of p53............... 33
Fig. 8. Mutation of P3 (372-380) or P2 (357-370) severely diminishes
the TBP-p53 interaction........................................................................................35
Fig. 9. Comparison of the TBP-binding capacities between the intact
C-terminus of p53 and the truncated forms by coprecipitation.......................... 36
Fig. 10. The transient transfection-transcription assay to evaluate
the effects of mutation of P5 (381-393) and (372-380) on the
transcriptional activation function of p53............................................................38-39
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Chapter 3
Fig. 1. Identification of the interactions of p53 and the deletion mutant
1-292 with human cyclin D l, D2, and D3 by coimmunoprecipitation...............64
Fig. 2. Activating effect by deletion of either the C- or the
N-terminus p53 on the p53-cyclin D interaction................................................. 65
Fig. 3. Determination of the cyclin D-binding domains in p53 by
Coimmunoprecipitation........................................................................................ 67-68
Fig. 4. Summary of the mapping results for cyclin D-binding
domains in p53 from Fig. 3 (A, B, and C) and unshown data.............................69
Fig. 5. EMSA to examine the DNA-binding activities of truncated
p53 proteins...........................................................................................................71-72
Fig. 6. EMSA for testing the formation of the DNA-truncated
p53-cyclin D tripartite complex........................................................................... 73
Fig. 7. Reciprocal exclusion between RB and the truncated p53
in the interaction with cyclin D3..........................................................................75
Chapter 4
Fig. 1. Localization of RBP2-binding domains in p53........................................91
Fig. 2. Mutation at P3 (372-380) or P2 (357-370) abrogates
the interaction between p53 and the C-terminus of RBP2.................................. 92
Fig. 3. Binding the RBP2 C-terminus 1200-1722 by p53 is
blocked by anti-p53 P3 antibodies PAb421 and PAbl22................................... 94
Fig. 4. Localization of p53-binding domains in RBP2........................................95
Fig. 5. The C-terminus of RBP2 binds double-stranded DNA
in a sequence-non-specific manner...................................................................... 97
Fig. 6. Competition for binding the C-terminus of RBP2
between 3 2 P-labeled and cold DNA fragments with same
or different sequences...........................................................................................98
Fig. 7. Schematic presentation of RBP2 deletion mutants
and the corresponding DNA-binding activities...................................................99
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Fig. 8. The truncated RBP2 1497-1722 does not stimulate the
sequence-specific DNA-binding activity of p53....................................................101
Fig. 9. Coexpression of RBP2 does not change the transcriptional
transactivity of p53................................................................................................... 102
Fig. 10. Coexpression of RBP2 does not affect the transcriptional
repression elicited by p53......................................................................................... 103
Fig. 11. The GAL4-RBP2 and GAL40-RBP2 C-terminus fusion
proteins can not enhance tmascription from the GAL4 binding
sites containing promoter.......................................................................................... 105
Fig. 12. Identification and localization of RBP2 and the truncated
mutant in transfected H358 cells by immunohistochemistry..................................107
Fig. 13. Comparison of the potentials of colony formation in H358
cells transfected with the wild-type RBP2 cDNA and in the same cells
transfected with the truncated mutant by immunohistochemistry.......................... 108
Fig. 14. The potential functional domains of RBP2.................................................112
TABLES
Chapter 4
Table 1. Structural information of pGEX2T expression vectors carrying
truncated RBP2 sequences........................................................................................ 115
Table 2. Cloning sites for the plasmids used for transient transfection-
transcription assay.................................................................................................... 116
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ABSTRACT
The C-terminus of p53 functions as a negative regulator for sequence-specific DNA
binding. Modifications of p53, such as binding by antibody PAb421 and phosphorylation
by several different kinases, overcome this regulation. However, which sequences at the
C-terminus participate in the activation process and how they coordinate to cause
conformational change are not completely clear. In order to answer these questions, we
studied the functions of the domain P5 (residues 381-393) and P3 (residues 372-380) in
p53, which are homologous to certain sequences in th e retinoblastoma susceptibility gene
product (RB) and are located outside the main functional domains of p53. Our studies
revealed that P5 and P3 participate in the regulation of p53. Deletion of P5, or mutation
of the P5 amino acid residues conserved between RB and p53, prevents p53 from being
phosphorylated by cyclin A/Cdk2 and activated by PAb421 . Mutation of P3 constitutively
activates p53 for sequence-specific DNA binding but severely decreases its ability to
interact with TBP. Deletion of P3 abrogates the binding of the C-terminal domain of p53
to TBP.
We have discovered several p53-binding cellular proteins while searching for
candidates that regulate the function of p53. Three of these are D-type cyclins D l, D2,
and D3 which interact most avidly with several truncated forms of p53. Two cyclin D-
binding domains in p53 were localized within the central core region, overlapping the
sequence-specific DNA-binding domain. The p53-cyclin D complex apparently lacks the
ability to bind the consensus p53 responsive DNA element. Truncated p53, cyclin D, and
RB proteins weakly form a tripartite complex.
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Another p53-binding protein is RB binding protein-2 (RBP2). Two p53-binding
domains in RBP2 and three RBP2-binding domains in p53 were defined. Mutation of P3
completely eliminates the RBP2-binding activity of p53.
Additional experiments on RBP2 function confirmed the importance of several newly
described protein-protein or protein-DNA binding domains: H358 tumor cells formed
fewer and smaller colonies when transfected with wild-type RBP2 cDNA than with a
RBP2 mutant carrying deletion of the overlapping region among these putative functional
domains.
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CHAPTER 1: BACKGROUND AND SIGNIFICANCE
p53 Is a Tumor Suppressor Gene
p53 was originally identified in a complex associated with the SV40 large T antigen, a
viral oncoprotein, from simian virus 40 (SV40) transformed mouse cells (Lane and
Crawford, 1979; Linzer and Levine, 1979). It was later defined as a tumor suppressor
gene based on the following critical discoveries: 1) A high frequency of p53 gene
mutations occurs in major forms of human cancers (Baker et al., 1989; Nigro et al., 1989;
Hollstein et al., 1991); 2) The Li-Fraumeni tumor-prone families carry germline
mutations of p53 (Malkin et al., 1990; Srivastava et al., 1990); 3) When introduced into
cells, the human wild-type p53 gene suppresses tumor cell growth (Baker et al., 1990;
Chen et al., 1990; Diller et al., 1990; Michalovitz et al., 1990; Takahashi et al., 1992) and
the murine wild-type p53 gene inhibits transformation of rat primary embryo fibroblasts
by ras plus adenovirus E1A, myc or mutant p53 (Finlay et al., 1989; Eliyahu et al., 1989).
The gene of human p53 is 20 kb long and has 11 exons (Lamb and Crawford, 1986).
It is located within chromosome 17 at 17pl2 to 17pl3.3 which is a common region of
allelic deletion found in more than 75% of colorectal carcinomas and in other types of
tumors, such as those of the brain, breast, lung, and bone. The remaining p53 allele in
these tumors contain a missense mutation (Baker et al., 1989, and the references therein).
Human p53 cDNA was isolated as a 2.95 kb fragment with an open reading frame of
1179 bases coding for 393 amino acid residues (Lamb and Crawford, 1986).
The fact that p53-deficient (-/-) mice develop normally indicates that p53 is not
essential for cell division and embryonic development. On the other hand, the
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susceptibility of these mice to a variety of tumors is consistent with the observation that a
high frequency of p53 mutation occurs in human cancers (Donehower et al., 1992). In
contrast to normal embryo fibroblasts, early passage embryonic fibroblasts derived from
53 (-/-) embryos are capable of gene amplification (Livingstone et al., 1992; Yin et al.,
1992) and became highly aneuploid after continued passage in culture (Yin et al., 1992).
Treatment of cells with UV and other DNA damaging reagents causes an increase in p53
protein level (Maltzman and Czyzyk, 1984; Kastan et al., 1991) and temporary G1 arrest
(Kastan et al., 1991). Based on these and other discoveries, such as the association of p53
with and the inactivation by DNA tumor viral oncoproteins SV40 large T antigen (Lane
and Crawford, 1979; Linzer and Levine, 1979; Bargonetti et al., 1991), adenovirus E1B
(Samow et al., 1982; Yew and Berk, 1992) and human papillomavirus E6 (Scheffner et
al., 1990; Werness et al., 1990), the genetic instability of fibroblasts from Li-Fraumeni
patients (Bischoff et al., 1990a), and the potential of wild type 53 to induce apoptosis
(Yonish-Rouach et al., 1991), a model concerning the function of p53 was proposed by
Lane and Kastan. Specifically, they propose that p53 acts as a molecular guardian for
genomic integrity. Its accumulation results in cessation of replication to allow repair in
response to DNA damage, or triggering of cell suicide if repair fails (Lane, 1992; Kastan
et al., 1992).
Function of p53 as a Transcription Activator
Accumulated evidence from the study of cellular effects elicited by p53 provided
supportive evidence for the model: that it acts as a cell cycle check point molecule and a
mediator of programmed cell death in response to DNA damage. The transcriptional
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activating function of p53 has been shown to be a major component of its effects (Crook
et al., 1994; Pietenpol et al., 1994). The mechanisms of p53-dependent G1 arrest and
apoptosis involve transcriptional transactivation by p53 of certain genes which function
as downstream effectors (Kastan et al., 1992; El-Deiry et al., 1993; Miyashita et al.,
1994a; Miyashita and Reed 1995). These genes include p21/WAFl/Cipl, which encodes
a cell growth suppression mediator and cell cycle-dependent kinase inhibitor (El-
lUnexpected End of FormuIaDeiry et al., 1993; Gu, Y. et al., 1993; Harper et al., 1993;
Xiong et al., 1993), GADD45, which encodes a S phase entry inhibitor (Kastan et al.,
1992; Smith et al., 1994), and bax, which encodes a protein promoting apoptosis (Oltvai
et al., 1993).
p21 works in at least two ways as a downstream effector of the growth control
pathway of p53. It associates with various complexes of G1 cyclin and G1 cyclin-
dependent kinase as a potent inhibitor, preventing the product of retinoblastoma
susceptibility gene (RB) from being phosphorylated (Harper et al., 1993; Xiong et al,
1993). This maintains RB in an active form which can bind to and inhibit the activities of
the cell cycle progression transcription factor E2F-1 responsible for activation of
transcription of multiple growth-related genes (described in detail in later section) needed
to drive quiescent cells into S phase (Nevins, 1992; Johnson et al., 1993). p21 also
directly inhibits DNA replication by binding to PCNA and interrupting its activation of
DNA polymerase 8 (Waga et al., 1994). Like p21, ectopic expression of GADD45 results
in growth arrest (Zhan et al., 1994; El-Deiry et al., 1994). GADD45 also associates with
PCNA, but unlike p21 (Xiong et al., 1992a), its presence is not detected in cyclin-Cdk-
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PCNA complexes, nor can it inhibit the cyclin E/Cdk2 dependent activity. Growth arrest
elicited by GADD45 may be pursued by regulation of PCNA functions in replication and
DNA repair through direct binding to this protein (Smith et al., 1994). Bax, a member of
Bcl-2 family, works downstream of the p53 transcription-dependent apoptosis pathway.
The mechanism by which it promotes cell death may involve its heterodimerization with
the death inhibitor Bcl-2 (Oltvai et al., 1993). However, the biochemical events leading to
cell death from this complex formation have not been well established.
Functional Domains Responsible for Transcription Activation of p53
The functional domains of p53 responsible for transcriptional activation have been
well documented. The sequence-specific DNA-binding domain (Bargonetti et al., 1993;
Pavletich et al., 1993; Wang et al., 1993) lies within the central core region (100-300).
This domain recognizes two copies of a consensus DNA sequence 5’-
RRRC(A/T)(T/A)GYYY-3’ and thereby determines the activating specificity of target
genes. The consensus sequence has been identified in either the promoter regions or
introns of a number of genes, including muscle-specific creatine kinase (MCK) (Zambetti
et al., 1992), GADD45 (Kastan et al., 1992), p21 (El-Deiry et al., 1993), mdm2 (Wu et
al., 1993), bax (Miyshita and Reed, 1995), IGF-BP3 (Buckblinder et al., 1995), cyclin G
(Okamoto and Beach, 1994; Zauberman et al., 1995) etc. Transcription of the endogenous
genes listed above is stimulated by ectopic expression of wild-type p53 or by induction of
the wild-type activity of temperature sensitive mutants (Kastan et al., 1992; El-Deiry et
al., 1993; Wu et al., 1993; Barak et al., 1993; Miyashita et al., 1994a; Okamoto and
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Beach, 1994; Selvakumaran et al., 1994; Buckblinder et al., 1995; Zauberman et al.,
1995).
Analysis of the p53 mutational spectrum in human tumors and tumor cell lines led to
the discovery that most of the hot spots of missense mutations are clustered within the
DNA-binding domain of the protein (Hollstein et al., 1991; Hollstein et al., 1994).
Tumor-derived p53 mutants (Vall43->Alal43, Argl75->Hisl75, Arg248->Trp248, and
Arg273->His273) fail to bind the consensus DNA elements (Kern et al., 1991; Kern et al.,
1992), to transactivate a reporter (Farmer et al., 1992; Pietenpol et al., 1994) or to
suppress tumor cell growth (Pietenpol et al., 1994). These facts together strongly argue
for the importance of DNA-binding capacity and transcriptional transactivity to the
biological function of p53.
The amino terminus of p53 (aa 1-43) contains the acidic activation domain (Fields
and Jang, 1990; Raycroft et al., 1990; Unger et al. 1992) which interacts with TATA-
binding protein (TBP) a component of the general transcription factor TFDD (Truant et
al., 1993; Liu et al., 1993; Martin et al., 1993) and its associated factors (TAFns) (Lu and
Levine 1995; Thut et al., 1995). The activation domain also interacts with p62, a subunit
of the general transcription factor TFIIH (Xiao et al., 1994; Wang et al., 1995). The
correlation between a transcription activating defect of the double point mutation mutant
p53 (Leu22->Glu22 and Trp23->Ser23) and its inability to bind T A F jj60 and T A F ji40
suggests that p53 may transmit the activation signal through the target coactivators
T A F jj6 0-TAFn40 to the basal transcription machinery (Thut et al., 1995). However, there
is no direct proof for the functional significance of the p53-TBP and p53-p62 interactions.
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Examination of other transcription activators has provided indirect evidence for the
relevance of p53-TBP interaction to transcriptional activation. Mutation that reduces both
binding activity to TBP and transactivity was found in all cases of the transcription
factors human T-cell leukemia virus type 1 Taxi (Caron et al., 1993), herpes simplex
virus VP16 (Ingles et al., 1991), human immunodeficiency virus type 1 Tat (Kashanchi et
al., 1994), and adenovirus E1A (Lee et al., 1991). This suggests that p53-TBP interaction
may accelerate assembly of the preinitiation complex on the promoters of genes
transcribed by RNA polymerase n. The finding that point mutations in VP 16 which
reduce TFIIH binding also reduce its transactivity (Xiao et al., 1994) implies that p53 may
also be involved at a later step in transcription, such as open-complex formation or chain
elongation.
The oligomerization domain (325-356) (Sturzbecher et al., 1992; Pavletich et al.,
1993; Wang et al., 1993; Jeffrey et al., 1995), the second binding domain to TBP
(Horikoshi et al., 1995) and the second binding domain to XPB and XPD components of
TFIIH (Xiao et al., 1994; Wang et al., 1995) reside within the C-terminus (300-393) of
p53. The oligomerization domain is required for the transactivity of p53 in some
experiments (Halazonetis et al., 1993; Pietenpol et al., 1994) but is dispensable in others
(Shaulian et al., 1993; Slingerland et al., 1993; Tarunina and Jenkins, 1993). It is possible
that this domain is necessary for binding of p53 to certain responsive elements but not
others. The relevance of binding of TBP and TFIIH by the C-terminus to the
transcriptional activation function of p53 is unclear.
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Unlike the specific DNA-binding domain, the activation and oligomerization domains do
not contribute to target specificity of p53. They can be replaced with other transactivation
domain and dimerization domain and still retain p53 transactivity without affecting
activation specificity (Pietenpol et al., 1994).
Transactivation-Independent Functions of p53
Further studies suggest that, besides transcription activation, other transactivation-
independent functions of p53 contribute to its cellular effects. For example, the
transcription defective mutants del214, containing only the first 214 amino acids of
murine p53, and p53/22, 23, which carries missense mutations in amino acids 22 and 23
of human p53, still act as potent inducers of apoptosis. The del214 mutant has also been
shown to suppress the transformation of rat fibroblasts by several oncogene combinations
(Haupt et al., 1995). The transactivation defective mutant p53A43 retains some ability to
suppress oncogene-mediated (ras plus El A) transformation of primary rat embryo
fibroblasts (Unger et al., 1993). The transcription defective murine p53 mutant tsp53/25-
26, carrying mutations in amino acid residues 25 and 26, can arrest cells at GO->S
transition in cooperation with Gasl (Schneider et al., 1988; Del Sal et al., 1995). In
addition, p53-dependent apoptosis can occur in the absence of transcriptional activation
of p53-targeted genes (Caelles et al., 1994).
A Putative Role for p53 in Regulation of Replication
Existing evidence suggests that p53 may play a role in the direct regulation of DNA
replication. For example, wild-type p53 can inhibit SV40 replication in vitro (Wang et al.,
1989; Friedman et al., 1990) and in vivo (Braithwaite et al., 1987). The C-terminus 30
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amino acid residues-truncated p53 can block Xenopus sperm nuclear DNA synthesis in
Xenopus egg extracts (Cox et al., 1995), while wild-type p53 inhibits replication of DNA
carrying the polyoma DNA virus replication origin and multiple copies of the p53 binding
site derived from the human genomic ribosomal gene cluster (RGC) in murine fibroblast
extracts (Kern et al., 1991; Miller et al., 1995).
Transcriptional Repression by p53
p53 represses transcription of a number of genes whose regulatory elements contain
TATA boxes but are devoided of consensus p53-binding sites. These include c-fos, c-jun,
IL-6, Rb, and bcl2 (Donehower and Bradley, 1993, and references therein; Mack et al.,
1993; Miyashita et al., 1994b). The data suggest that the mechanism of repression
involves direct binding and sequestering TBP and/or its associated factors (TAFjjs) by
p53, inhibiting transcription initiation (Seto, E. et al., 1992; Mack et al., 1993; Horikoshi
et al., 1995; Shaulian et al., 1995; Sabbatini et al 1995a). Since the genes transcriptionally
repressed by p53 are involved in either growth regulation or apoptosis, and the adenovirus
E1B 19-kD protein and the cellular protein Bcl-2 both block p53-dependent apoptosis by
suppressing its transcriptional repression activity (Shen and Shenk, 1994; Sabbatini et al.
1995b), the transcriptional repression function of p53 is considered to also play a role in
apoptosis and tumor suppression.
p53 also represses transcription of the hsp70 promoter by direct interaction with and
sequestration of the transcriptional activator CBF (Agoff et al., 1993).
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Regulation of p53 in Cells
Since p53 elicits a potent effect on the life and death of cells, it is reasonable to
believe that the level and the activity of p53 are tightly and precisely controlled. Indeed,
multiple pathways have been demonstrated which indicate that the activity of p53 is
regulated at the transcriptional, translational and post-translational levels.
Wild-type p53 is present at an extremely low level and has a very rapid turnover rate,
on the order of minutes (15-25 min), in most cells. Expression of p53 is regulated both
during the cell cycle and in emergency response. During the cell cycle, mRNA and
protein levels of p53 increase upon enter to G1 from G O , and reach maximum in late G1
(Milner and McCormick, 1980; Milner and Milner, 1981; Reich and Levine, 1984). In
response to genotoxic stress, the protein concentration increases significantly and the
elevation is detected at both the post-transcriptional (Kastan et al., 1991), and
translational levels (Fritsche et al., 1992).
Transcription of the p53 gene is stimulated by the p53 protein itself. This regulation is
pursued through the p53 responsive element (+22 to +67) within the p53 promoter,
although a direct p53-DNA interaction can not be detected in vitro (Deffie et al., 1993).
Results from in vitro translation experiments suggest that the RNA binding and re
annealing capacity of p53 may contribute to the regulation of p53 mRNA translation. p53
tightly binds the 5’-UTR and inhibits translation of its own mRNA, which has a predicted
tendency to form a stable stem-loop structure between the 5’-UTR and the downstream
280 nucleotides. Furthermore, this translation inhibition is substrate selective (Mosner et
al., 1995).
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Besides regulation at the level of protein expression, the activity of p53 as a
transcriptional activator may also be regulated throughout the cell cycle. Evidence
supporting this hypothesis comes from the observation that transfected p53 does not
activate its responsive element containing promoter in the GO phase unless cotransfected
with cyclin E (Deffie et al., 1995), a late G1 cyclin. Furthermore, a coincident elevation
of sequence-specific DNA-binding activity but protein level indicates modulation of
DNA binding activity of p53 as the mechanism (Deffie et al., 1995). However, since p53
is not the substrate of the cyclin E-Cdk2 complex (Wang and Prives, 1995), the activation
effect must be indirect.
The ability of p53 to activate transcription is negatively regulated by mdm2 via a
feedback loop (Wu et al., 1993). The mdm2-binding domain (1-52) overlaps with the
activation domain of p53 (1-43) (Oliner et al., 1993). The mechanism of inhibition may
involve precluding the interactions of p53 with TAFj j 40 or TAFn60 (Thut et al., 1995)
and with the consensus DNA element (Zauberman et al., 1993).
Phosphorylation may also enhance the ability of p53 to activate transcription. There
are multiple phosphorylation sites, distributed throughout the the p53 sequence, for
various kinases, such as the cyclin-dependent kinases (Cdks) (Addison et al., 1990;
Bischoff et al., 1990; Wang and Prives, 1995), casein kinases I (CK-I) (Milne et al., 1992)
and II (CK-EI) (Meek et al., 1990), double-stranded DNA-activated protein kinase (DNA-
PK) (Lees-Miller et al., 1990; Lees-Miller et al., 1992; Wang and Eckhart, 1992) and
protein kinase C (PKC) (Baudier et al., 1992). Phosphorylation by CK-II (Hupp et al.,
10
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1992), PKC (Hupp and Lane, 1994; Takenaka et al., 1995) and the Cdks (Wang and
Prives, 1005) can activate the sequence-specific DNA binding of p53 in vitro.
Mechanisms of Activation of p53 Transactivity
Previous studies have provided evidence that the sequence-specific DNA-binding
capacity is crucial for the transcriptional activation function of p53 (Kern et al., 1991;
Farmer et al., 1992; Deffie et al., 1995; Zauberman et al., 1995). Since p53 is not
constitutively active in sequence-specific DNA binding, some mechanism(s) must exist
by which the binding potential of latent p53 is activated. Accumulated experimental
results suggest possible mechanisms by which the latent p53 is activated.
Recombinant p53 protein expressed in bacterial cells is incapable of binding DNA
unless its carboxyl terminus is bound by the monoclonal antibody PAb421 at epitope 371-
380 or by bacterial heat shock protein DnaK (Hupp et al., 1992). Activation of p53 can
also be achieved by the deletion of amino acid residues 364-393 (Hupp et al., 1992), the
addition of short single strands of DNA (Jayaraman and Prives, 1995), or the
phosphorylation of Ser392 by CK-II (Hupp et al., 1992), Ser378 by PKC (Hupp and Lane,
1994; Takenaka et al., 1995), or Ser315 by Cdks (Wang and Prives 1995).
Based on these observations, a model for the activation mechanism was proposed by
Lane and colleagues: a negative regulatory domain lies within the C-terminal 30 amino
acid residues (364-393), which maintains the molecule in the latent state for sequence-
specific DNA binding. Some molecular event(s), either modification or a protein
interaction occurring in/around this sequence, changes the conformation of p53,
switching it from the inactive to the active form. By the use of short peptides (370-382
11
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and several overlapping sequences), the same group proved and further developed this
model. The fact that addition of the specific short peptides to CK-H phosphorylated p53
proteins at 0°C can activate sequence-specific DNA binding suggests that sequence 370-
382 plays a role in allosterically regulating conformation, but does not block the DNA
binding domain as a steric inhibitor (Hupp et al., 1995). The authors suspected that
residues 370-382 interact with the core region of the same or another p53 molecule to
retain the tetramer in the latent state, and that posttranslational modification at the
regulatory site can disrupt this interaction which leads to dissociation of the DNA binding
inhibiting residue-residue interaction. The resulting new conformation facilitates p53-
DNA interaction.
In support of the proposed model, microinjection of PAb421, which binds to the C-
terminal sequence 372-378, was shown to increase transactivity of p53 in cells (Abarzua
et al., 1995; Hupp et al., 1995).
It was also reported that incubation with TBP increases the DNA binding activity of
p53 (Chen et al., 1993). Since TBP physically interacts with both the N- and C-termini of
p53, it is possible that the activation mechanism involves a conformational change caused
by a physical interaction between the carboxyl-terminus of p53 and TBP.
RB and p53 Share Homologous Sequences
The retinoblastoma susceptibility gene (Rb) is another tumor suppressor gene which
has been cloned by our lab and other groups (Friend et al., 1986; Fung et al., 1987; Lee et
al., 1987). The RB protein is a transcription regulator (for review, see Weinberg, 1995,
and Seller and Kaelin, 1996), crucial for tumor suppression (Huang et al., 1988), embryo
12
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development (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992), and cell terminal
differentiation (reviewed by Chen et al., 1995). In our laboratory, we made the discovery
that anti-RB polyclonal antibodies Ab-18 and Ab-20 can also recognize p53. Visual
comparison of the protein sequences revealed the existence of homology between RB
(871-886) and p53 (3 81-393) in a short stretch of sequence designated P5, as shown in
Fig. 1. The recognition of p53 blocked by the addition of P5 peptide further confirmed the
antigenic homology of these two proteins at P5. A further search for other homologous
pairs of peptides, using a computer alignment program, revealed these homologous
sequences P3, in addition to P5. Both P5 and P3 reside outside the central core regions of
RB (379-792) and p53 (100-292) (Fig. 1). In p53, P5 (381-393) and P3 (372-380) are
located within the carboxyl-terminus, which contains the negative regulatory domain for
sequence-specific DNA binding (Hupp et al., 1992). These facts suggest a possible
involvement of P5 and P3 in conformational regulation.
Study of RB/p53 Homologous Sequences-Mediated Functional
Regulation of p53 in Our Laboratory
Our laboratory has been working on determining the role of the homologous
sequences P5 and P3 in the functional regulation of RB and p53. Chapter 2 of this thesis
describes hypotheses and research progress in this area, with emphasis on the effect of
mutation of the RB/p53 homologous sequences P5 and P3 in p53 on its phosphorylation
by Cdk2, its sequence-specific DNA binding activity, and its TBP binding capacities.
Expanding from the research in Chapter 2, Chapters 3 and 4 of the thesis describe
respectively of the discovery of the physical interactions of cyclin D and RB binding
13
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Ser807 Ser811
LT binding
DNA. binding
Ser315
P)
P3P5
p53
393
RB P5(873-886) : KKLRFDIEG— SD
p53 P5(381-393): KKLMFKTEGPDSD
RB P3(245-262): RRGONRSAR
p53 P3(372-380): KKGOSTSRH
Fig. 1. Homologous sequences P5 and P3 between RB and p53.
Areas corresponding to peptides P5 and P3 in RB and p53 are
filled and shaded with black and white squares resdpactively. Two
Cdk sites controlled by P5 in RB are indicated as P in circles, so is
the Cdk site in p53. Amino acid residues and their positions for
phosphorylation are labeled above the circles. Amino acid
residues conserved between the two proteins, within P5 and P3,
are underlined
14
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protein-2 (RBP2) with p53, their binding profiles, and our progress in determining their
functional relationship with p53.
15
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CHAPTER 2: THE CARBOXYL-TERMINUS OF p53 REGULATES TIS DNA
BINDING AND KINASE REACTION
Summary
Tumor suppressors RB and p53 are both phosphorylated by cyclin-dependent kinases
(Cdks). Phosphorylation by Cdks is believed to be the main regulatory mechanism by
which the function of RB is inactivated. Phosphorylation of p53 by Cdks enhances its
sequence-specific DNA binding activity in vitro, although the biological significance of
this modification is unclear. RB and p53 share homology at two small regions, P5 and P3,
which lie within the assumed regulatory domains of these two proteins. The functions of
the sequences are unknown. Our laboratory has initiated a study of the roles P5 and P3
play in the functional regulation of both RB and p53. Our laboratory discovered that
mutation of P5 (873-886) prohibited phosphorylation of RB at several Cdk sites, which
are believed important for control of conformation and activity of the RB protein.
Analysis of phosphorylation of P5 missense and deletion mutants of p53, by the same
assay, shows that mutation or deletion of P5 completely inhibits phosphorylation on
Ser3i5 by Cdk2. This observation indicates a conformational change of the p53 mutant,
which may impact on its other properties. Examination of DNA binding capacities of P5
missense and deletion mutants, using the gel electrophoresis mobility shift assay
(EMSA), shows that mutation or deletion of P5 completely abrogates the binding ability
of p53. In addition, since elimination of 30 amino acid residues from the carboxyl-
terminus of p53 is reported to relieve the constraint on the sequence-specific DNA
binding activity (Hupp et al., 1992), and P3 (372-380) lies within this region, we also
16
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tested a P3 missense mutant for its DNA binding capacity by EMSA. Mutation at P3
causes p53 to become constitutively active for DNA binding. This is consistent with the
result of a study by Lane and colleagues (Hupp et al., 1995).
While studying P3 function, we became aware that TATA-binding protein (TBP), a
subunit of transcription factor TFIID, binds both the amino- and the carboxyl-termini of
p53 (Seto, E. et al., 1992; Truant et al., 1993; Liu et al., 1993; Martin et al., 1993;
Horikoshi et al., 1995) and that it can stimulate p53 sequence-specific DNA binding
activity (Chen et al., 1993). We suspect that the mechanism by which TBP stimulates this
activity might involve regulation of p53 conformation through binding to P3 and its
vicinity. Comparison of the effects of mutation at P3 and proximal sequences, on p53-
TBP interaction by coimmunoprecipitation, shows that mutation of P3 has the strongest
negative effect on the binding activity of p53 among all the C-terminal mutations tested.
More detailed mapping of the carboxyl-terminal TBP-binding domain shows that amino
acid sequences 293-393, 293-383, and 325-393 have a similar affinity for p53. The
sequence 357-393, which contains the minimal TBP-binding activity, includes P3 (372-
380) and the proximal sequence 357-370, which coincides with the regulatory motif (357-
380) for sequence-specific DNA binding defined by EMSA. These results indicate the
importance of P3 for TBP binding, and imply that the mechanism of the DNA-binding
activation mediated by TBP may involve a TBP-P3 interaction and a thereby-induced
conformational change.
To find out if P5 and P3 mediate the transcriptional regulation function of p53, we
examined the effects of mutation/deletion of these two sequences on the transcriptional
17
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transactivity of p53 in H358 cells (a non-small cell lung carcinoma cell line which is p53
-/-) using a transient transfection-transcription assay. No difference between wild type
and the P5 mutation/deletion (381-393) mutants can be detected under our experimental
conditions. Negligible or slight transcription stimulating effects are elicited by the P3
(372-380) missense mutant and a P5 plus P3 (367-393) deletion mutant when compared
with wild type. These observations suggest that multiple activating mechanisms may exist
in vivo, which mask the individual contributions of the investigated factors in the
regulation of DNA-binding dependent transcriptional activation.
Introduction
Demonstrated Regulatory Functions of P5 and P3
The retinoblastoma susceptibility gene product (RB) is a phosphoprotein the function
of which is believed to be regulated through phosphorylation in a cell cycle dependent
manner by Cdks (Buchkovich et al., 1989; Chen et al., 1989; DeCaprio et al., 1989;
Mihara et al., 1989). As introduced in Chapter 1, RB and p53 share homology within
some small regions. Two of them are designated P5 and P3. These two homologous
sequences are located outside the main functional domains of the both proteins (Fig. 1).
To determine whether P5 plays a role in the regulation of RB function, our laboratory
examined the P5 mutant of RB with respect to conformation and phosphorylation pattern
change in comparison with wild type. Mutation of P5 (873-886) leads to a conformational
change in RB protein such that two Cdk sites, S ergo 7 and Sergn, are refractory to
phosphorylation both in vitro and in vivo (Driscoll et al., submitted to J. Biol. Chem.).
18
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Underphosphorylated S e rg o ? and Sergn were also observed in RB protein extracted from
TGF-B treated, growth arrested, cells (Driscoll et al., 1995).
Amino acid residues 370-382, nearly equivalent to P3 (372-380), was reported
recently by Lane and colleagues to be a key sequence directly responsible for maintenance
of p53 in the inactive state for sequence-specific DNA binding (Hupp et al., 1995).
Further Questions Raised and Research Progress
Following the demonstration of a function for P5 in the RB protein, we investigated
whether P5 in p53 plays the same role, since p53 is also a reported substrate of Cdk2 and
Cdc2 (Bischoff et al., 1990; Wang and Prives, 1995). In addition, since P5 (381-393) lies
within the carboxyl-terminus of p53, known to be a regulatory domain for sequence-
specific DNA binding, w e suspected that it might play a role in control of DNA-binding
activity. Furthermore, w e were also interested to know, other than the kinases CK-II,
PKC, and Cdks, which can phosphorylate and activate p53 for DNA binding (Hupp et al.,
1992; Hupp and Lane, 1994; Takenaka et al, 1995; Wang and Prives, 1995), whether
another type of cellular factor can non-covalently modify the C-terminus of p53 by
physical interaction, thereby converting the conformation from an inactive to an active
form for sequence-specific DNA binding.
Our research has yielded several findings: 1) As with RB, P5 in p53 contributes to the
formation of a conformation in which Cdk2 is able to recognize and phosphorylate Ser3is.
2) P5, as opposed to P3 (372-380), participates in the activation process modulated by
PAb421. 3) P3 is absolutely required for the interaction between p53 and TBP.
19
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RB
P3
I
LT,E1A and E7
binding
A B
Ser807 Ser811
928
Ser315
LT bindingOD
DNA binding P3P5
p53
393
RB P5(873-886):
p53 P5(381-393):
RB P3(245-262):
p53 P3(372-380):
KKLRFDIEG— SD
KKLMFKTEGPDSD
RRGONRSAR
KKGOSTSRH
Fig. 1. Homologous sequences P5 and P3 between RB and p53.
Areas corresponding to peptides P5 and P3 in RB and p53 are
filled and shaded with black and white squares resdpactively.
Two Cdk sites controlled by P5 in RB are indicated as P in circles,
so is the Cdk site in p53. Amino acid residues and their positions
for phosphorylation are labeled above the circles. Amino acid
residues conserved between the two proteins, within P5 and P3,
are underlined
20
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Results
Mutation or Deletion of P5 Alone But the Other Surrounding Sequences Inhibits
Phosphorylation of the p53 Protein on Ser3is
There is a single Cdk site at Ser3i5, in human p53 (Bischoff et al., 1990b; Addison et
al., 1990) the phosphorylation of which by Cdk2 and Cdc2 activates the sequence-specific
DNA binding of p53 efficiently (Wang and Prives, 1995). To investigate the role played
by P5 in determining the conformation of p53 and phosphorylation of Ser3is, an in vitro
phosphorylation assay was performed (Fig. 2 and 3). The p53 mutants included mP5C
(conserved residues 381-383, 385, 388, 389, 392, and 393 within 381-393 mutated),
mP5U (non-conserved residues 384, 386, 387, 390, and 391 within 381-393 mutated),
mP3C (conserved residues 372-375, 378, and 380 within 372-380 mutated), mP3C’
(conserved residues 372-375 within 372-380 mutated), mPl (343-351 selectively
mutated), mP2 (357-370 selectively mutated), and various truncated forms 1-380, 1-366,
1-351, 1-342, 1-332, 1-318, 1-304, and 1-292. Structures of the missense mutants above
and in the later text are described in detail in Materials and Methods. Antibody/protein A-
agarose (PAA) bound p53 proteins prepared from bacteria were incubated with crude
cyclin A/Cdk2 (prepared from recombinant baculoviruses infected insect Sf9 cells) and
[y-3 2 P] ATP/cold ATP. Among all the tested missense mutations, only mutation at
conserved residues within P5 (381-393) but not at the unconserved residues, severely
inhibited phosphorylation of Ser3is by cyclin A/Cdk2 (Fig. 2A, left panel, lane 3, and 2B,
lane 2). The deletion mutant 1-380, like mP5, failed to be phosphorylated (Fig. 2A, left
panel, lane 5, Fig. 2B, lane 6, and Fig. 3, upper panel, lane 2), but mutants with deletions
21
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Fig. 2. Deletion of P5 (381-393) or mutation of conserved amino acid residues between
RB and p53 within P5 prohibits phosphorylation of p53 by Cdk2. In vitro
phosphorylation experiment was performed to investigate whether deletion/ mutation of
P5 or the vicinal sequences affects phosphorylation of Ser3is of p53 by Cdk2. mP5C (A,
lane 3, and B, lane 2) is the mutant carrying missense mutation at conserevd amino acid
residues within P5, mP5U (A, lane 4, and B, lane 7) carrying unconserved residue
missense mutation within P5, 1-380 (A, lane 5, and B, lane 6) with deletion of P5, mP3C
(B, lane 3) carrying missense mutation at conserved residues 372-375, 378 and 380
within P3 (372-380), mP3C' (B, lane 4) carrying missense mutation at conserved residues
372-375, and mP2 (B, lane 5) carrying selective mutation within P2 (357-370).
(A) Deletion of P5 or mutation of conserved, but not unconserved residues within P5
prohibits phosphorylation of p53 by Cdk2. The left panel is a phosphorylation
experiment, and the right panel is a immunoprecipitation experiment showing the
quantity of each protein used for phosphorylation in the left panel. Bacteria carrying the
p53 and its mutant expression plasmids were grown to log phase and divided into two
subcultures which were induced for protein expression in the absence and the presence of
L-[3 5 S]methionine and L-[3 5 S]cysteine respectively. The bacteria were aliquoted,
pelleted, and frozen at -80°C. First, immunopreci- pitation in the right panel was
conducted for testifying the protein expression and examining the protein quantities.
5xl08 35S-labeled frozen cells were lysed with 100 pi of PBS containing 0.25 mg/ml
lysozyme. 20 pi of hybridoma medium containing the anti-p53 monoclonal antibody
PAbl22 were mixed with 25 pi of the lysates as indicated and precipitated with PAA.
Since same number of bacteria from each sample showed a very similar protein quantity,
Each lysate prepared from the same number of bacteria was used later for the kinase
reaction in the left panel. 25 pi of the cold cell lysates as indicated were combined with
20 pi of the PAbl22 containing hybridoma culture medium and precipitated with PAA.
Impurities in the lysates were rinsed away with EBC solution. The purified protein-
antibody-PAA complexes were incubated with crude cyclin A/Cdk2 (prepared from
recombinant baculoviruses coinfected Sf9 cells) in the presence of cold and [y- P] ATP at
30°C for 1 hr. The sample in lane 1 in the left panel doesn't contain Cdk2 otherwise is as
same as lane 2. The unreacted ATP molecules were removed by rinsing the protein-
antibody-PAA complexes with EBC. The kinase treated proteins were resolved on 8%
SDS-PAGE as described in Materials and Methods.
(B) Only mutation of P5, but the other vicinal sequences tested affects phosporylation of
p53 by Cdk2. The kinase reaction was carried out as in (A) except that 0.2 pg of the anti-
p53 N-terminus monoclonal antibody DO-1 was used for precipitation of the p53 and
mutant proteins.
22
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cn O p o
■ g ^ v S C n o «
§ P< a . a , n
U
* 4 i V )
1 “ I
cn
u n
o o
in m 0 0
% %
c n
a a
1
PUP-
cn
u n
U O
*o cn
O
cn (N
i i i i
o
oo
D
m
1 2 3 4 5 6 7
Fig. 2. Deletion of P5 (381-393) or mutation of the conserved amino
acidresidues between RB and p53 within P5 prohibits phosphorylation of
p53 by cyclin A/Cdk2
23
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1 2 3 4 5 6 7 8 9
Fig. 3. Further deletion of p53 from residue 380 recovers the availability to be
phosphorylated on Ser3 1 5 by Cdk2. The in vitro phosphorylation experiment
with cyclin A/Cdk2 was conducted as described in Fig. 2A. The p53 and
truncated proteins were precipitated with 0.2 micro g of the antibody DO-1.
The upper panel: Phosphorylation of p53 and the mutants by Cdk2. 1-380 (lane
2) is a mutant with deletion of P5 (380-393), 1-366 (lane 3) with deletion of P5
plus P3 (372-380) and the partial of P2 (357-370), 1-304 (lane 8) with deletion
of the sequence including the Cdk2 site Ser3 1 5 , and the other mutants 1-351
(lane 4), 1-342 (lane 5), 1-332 (lane 6), and 1-318 (lane 7) with the C-terminus
truncated sizes between the mutants 1-366 and 1-304. The lower panel:
Immunoprecipitation for testing the protein expression and equalizing the
quantities of the proteins to be tested in (A). Bacterial cell numbers from
different samples were adjusted to provide same quantity of the proteins. The
photo shows the protein amount from each sample after adjustment. The same
quantity of the proteins were later used for the kinase reaction in (A).
24
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from 380 up to 332, i.e. 1-366, 1-351, 1-342, and 1-332 regained the ability to be
phosphorylated (Fig. 3, upper panel, lanes 3-6). These results together indicate that 381-
393 is not part of the sequence participating in the interaction between the substrate and
the enzyme, but instead is part of the sequence responsible for maintaining a
conformation in which Ser3i5 can be recognized and phosphorylated by Cyclin A/Cdk2.
The effect of binding by monoclonal antibody PAb421 on phosphorylation of Seijis
was also tested to find if P5 is the sole region responsible for maintaining the
conformation required for phosphorylation. Both Pab421 and PAbl22 are anti-p53 C-
terminus monoclonal antibodies with epitopes localized within residues 370-380. Given
that PAb421 can change the latent conformation of p53 into the DNA-binding active
form (Hupp et al, 1992), it is possible that binding by PAb421 may also inhibit the kinase
reaction, if P3 (372-380) is also responsible for maintenance of the conformation required
for phosphorylation by Cdk2. However, as shown in Fig. 4, both PAb421 (left panel, lane
1) and PAbl22 (left panel, lane 2) bound p53 proteins were heavily phosphorylated by
Cdk2, when compared with anti-N-terminus (epitope 21-25) monoclonal antibody DO-1
bound p53 (left panel, lane 3).
Mutation or Deletion of P5 Causes Failure of Sequence-Specific DNA-Binding
Activated by PAb421
To determine whether P5 (381-393) participates in the regulation of sequence-specific
DNA binding activity, EMSA was performed using wild-type p53, missense mutants
mP5C and mP5U, and truncated forms 1-380, 1-366, 1-351 and 1-342 (Fig. 5). Consistent
with the findings of Lane and colleagues (Hupp et al., 1992), wild type p53 doesn’t bind
25
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Cyclin A/Cdk2
421 122 DO-1 421 122 DO-1
m *# '
1 2 3 1 2 3
Fig. 4. Binding by anti-p53 C-terminus monoclonal antibodies PAb421 and
PAbl22 does not interfere with phosphorylation by Cdk2. Availibility of the
monoclonal antibodies PAb421, PAbl22, and DO-1 bound p53 protein to be
phosphorylated by Cdk2 in vitro were compared. The epitope recognized by
PAb421 is the C-terminal residues 371-380 (« P3, 372-380), and the epitope
recognized by DO-1 the N-terminal residues 21-25. Bacteria (in log phase)
carrying the p53 expression plasmids was divided into two subcultures which
were induced for protein expression in the absence and the presence of L-
[3 5 S]methionine and L-[3 3 S]cysteine. 5x10s cold bacterial cells were lysed with
100 pi of PBS containing 0.25 mg/ml lysozyme. The phosphorylation result is
shown in the left panel. 25 pi of the lysate were combined with 0.2 pg of
PAb421, PAbl22, and DO-1 respectively as indicated and precipitated with
PAA. Impurities in the lysate were rinsed away with EBC solution. The purified
protein-antibody-PAA complexes were incubated with crude cyclin A/Cdk2
(prepared from recombinant baculoviruses coinfected Sf9 cells) in the presence
of cold and [y-3 2 P]ATP at 30°C for 1 hr. The unreacted ATP molecules were
removed by rinsing the protein-antibody-PAA complexes with EBC. The kinase
treated proteins were resolved on 8% SDS-PAGE. The right panel is a control
experiment in which the quantities of the p53 protein combined by the three
antibodies were tested. 5x10s 3 : > S-labeled bacterial cells were lysed as in the
experiment on the left panel. 25 pi of the lysate were combined with 0.2 pg of
the three antibodies respectively as indicated and precipitated with PAA. The
antibody bound proteins were resolved on 8% SDS-PAGE.
26
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PAbl801 - - + + + +
PAb421 - + + - - + + - - + + -
wtp53 mP5U mP5C
mm mm m m < 4^
A
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 5. Deletion of P5 (381-393) or mutation on the conserved amino acid
residues between RB and p53 within P5 prohibits p53 from binding the
consensus DNA. DNA-binding capacities of the P5 and other mutants were
compared with wild type p53 by EMSA. 5x109 bacterial cells containing the wild
type and mutant p53 proteins were lysed with 200 pi of 50 mM HEPES
containing 10% sucrose and 0.5 mg/ml lysozyme. 0.1 pg of antibodies or the
equal volume of buffer was mixed and incubated with 1 pi of each lysate as
indicated at 0°C for 30 min, then 0.1 of the 32P-labeled p53 binding sites
containing DNA BC was added to each sample and continuously incubated for
another 30 min. The protein-DNA complexes were resolved on 4% PAGE.
(A) and (B) The p53 mutant with deletion of P5 or mutation of the conserved
amino acid residues within P5 fails to bind DNA in the absence and the presence
of activating antibodyPAb421. PAb421 was added to activate the latent p53 and
mutants for DNA binding. PAbl801 doesn’t have such function,, it was added to
supershift the PAb421-protein-DNA complexes. PAbl801 was also added in the
absence of PAb421 for providing the background for PAb421-induced sequence-
specific DNA binding signals. The epitopes recognized by PAb421 and PAbl801
are residues 371-380 and 45-55 respectively.
27
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Fig. 5 (continued)
PAbl801 - -
PAb421 - +
wtp53
- + +
+ + -
1-380
- + +
+ +
1-380
B
1 2 3 4 5 6 7 8 9 10
wtp53 1-342 1-351 1-366
PAbl801 - - + +
PAb421 -
1 2 3 4 5 6 7 8 9 10 11 12
(C) Further deletion from residue 380 recovers the DNA-binding activity of
p53. The p53 mutants with further deletion from residues 380 were also
tested for their DNA binding capacities. 0.1 pg of PAb421 and PAbl801
were respectively added to each of the mutants as indicated
28
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the consensus DNA “BC” (Halazonetis et al., 1993) unless it is activated by the addition
of PAb421 (Fig. 5A and 5B, lane 1 versus lane 2). mP5C, which carries mutations at the
P5 residues conserved between RB and p53, is unable to bind DNA, even with addition
of PAb421 Fig. 5A, lanes 9-11). mP5U, carrying mutations within the same region as
mP5C, but at unconserved residues, behaved in this assay identically to wild type
(compare Fig. 5A, lanes 5-8, with lanes 1-4). When the truncated forms 1-380, 1-366, 1-
351 and 1-342, are tested by the same assay, 1-380, with deletion of P5, like mP5C, fails
to bind DNA (Fig. 5B, lanes 3 and 7), even in the presence of PAb421 (lanes 4, 5, 8, and
9). However, upon deletion of 14 more amino acids from 380, the mutants 1-366, 1-351,
and 1-342 become active in the absence and presence of PAb421 (Fig. 5C, lanes 4-12).
The DNA-binding capacity of truncated p53 is maintained until deletion reached residue
292 (Chapter 3, Fig. 4B, lanes 9-11).
This observation implies a positive role for P5 (381-393) in the regulation of the
DNA-binding activity of p53. Also the P5 region appears to be specially required in order
for PAb421 to antagonize the negative effect exerted by P3. However, since the epitope
for PAb421 has been mapped to the sequence 371-380 adjacent to P5 (381-393), the
possibility exists that mutation or deletion of P5 may disturb the binding of mutants, mP5
and 1-380, by PAb421. To test this possibility, the abolity of pAb421 to
immunoprecipitate the mutants were tested (Fig. 6). Recombinant wild-type p53, mP5C,
mP5U, mP3C, mP3C\ mP2, and 1-380 expressed in bacteria were precipitated with three
anti-human p53 monoclonal antibodies DO-1 (or PAbl801), PAb421, and PAbl22. DO-1
(Fig. 6A, lanes 1, 4, 7, 10, and 13) and PAbl801 (Fig. 6B, lanes 1, 4, 7, 10, and 13), the
29
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Fig. 6. Immunoprecipitation of wild-type p53 and the mutants to examine whether
mutation or deletion of P5 (381-393) impairs the recognition of the protein by anti-P3
antibody PAb421. Structures of the p53 mutants are described in the text and Fig. 2.5xl08
bacterial cells containing the 3 5 S-labeled wild type and mutant p53 proteins were lysed
with 100 |il of PBS containing 0.25 mg/ml lysozyme. Three 25 pi aliquots from each
lysate were combined with 0.3 pg of three anti-p53 antibodies respectively and
precipitated with PAA. The precipitated proteins were resolved on 8% in (A) and 10%
SDS-PAGE in (B) as described in Materials and Methods.
(A) Among the mutations tested is only the mutation of P3 (372-380) that abrogates the
interactions between p53 and antibodies PAb421 and PAbl22. The antibodies DO-1,
PAb421, and PAbl22 were respectively added to each lysate containing the wild type or
mutant p53 protein as indicated. The epitopes for DO-1 and PAb421 were reported
(Oncogene Research Products) to be the N-terminal residues 21-25 and the C-terminal
residues 371-380 respectively. The experiment here showed that the epitope for PAbl22
is 370-380.
(B) Deletion and mutation of P5 do not affect recognition of p53 by PAb421. The
antibodies PAbl801, PAb421, and PAbl22 were respectively added to each lysate
containing the wild type or mutant p53 protein as indicated. The epitope for PAbl801 is
the N-terminal residues 46-55.
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30
DO-1 + - - + - - + - - +
P Ab421 - + - - + - - + - - + -
PAbl22 +
wtp53 mPl mP2 mP3C’ mP3C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
PAbl801 +
PAb421 -
PAbl22 - +
wtp53 mP5C mP5U 1-380 1-292
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Fig. 6. Immunoprecipitation of wild-type p53 and the mutants to examine
whether mutation or deletion of P5 (381-393) impairs the recognition of p53
by anti-P3 antibody PAb421
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
epitopes of which were mapped to the N-terminus of p53, recognize both wild type and
all mutant proteins. 1-292, which does not carry the epitopes for PAb421 and PAbl22, is
not recognized by these two antibodies (Fig. 6B, lanes 14 and 15). mP5C (lanes 5 and 6),
mP5U (lanes 8 and 9), 1-380 (lanes 11 and 12), mPl (Fig. 6A, lanes 5 and 6) and mP2
(lanes 8 and 9) are recognized by both PAb421 and PAbl22, with an affinity
indistinguishable from that of wild-type p53 (Fig. 6A and 6B, lanes 2 and 3). mP3C\
which is mutated at residues 372-375, is still recognized by PAb421, but not by PAbl22.
Only mP3C, which is mutated at residues 372-375, 378, and 380, is recognized by neither
PAb421 (Fig. 6A, lane 14) nor PAbl22 (lane 15).
Mutation at P3 or P2 Constitutively Activates p53 for DNA Binding
According to Lane et al. (Hupp et al., 1992), deletion of 30 amino acid residues from
the C-terminus of p53 activates p53 for sequence-specific DNA binding. Since P3 lies
within this motif, we wished to determine whether mutation of P3 alone can create the
same effect. We tested mP3C (conserved residues within 372-380 mutated) and several
other mutants, with missense mutations within the proximal sequences, including mPl
(343-351 selectively mutated) and mP2 (357-370 selectively mutated), by EMSA (Fig. 7).
Once again wild-type p53 doesn’t bind BC (Fig. 7, lane 1) unless PAb421 is added (lane
2). mP2 and mP3C exhibit a strikingly strong DNA binding activity in both the presence
(lanes 8 and 11) and absence of PAb421 (lanes 7, 9, 10, and 12). A considerable DNA-
binding activity, but an altered migration pattern, is observed for mPl in the absence
(lanes 4 and 6) and presence of PAb421 (lane 5). As described in the previous section, no
32
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PAbl 801 -
PAb421 - +
wtp53 mPl mP2 mP3C mP5C mP4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Fig. 7. EMSA for testing the effects of mutation of P3 (372-380) and P2
(357-370) on the sequence-specific DNA-binding activity of p53. Most of the
mutants examined in this experiments are described in the text and Fig. 2.
The mP4 protein added in lanes 16-18 was applied as a negative control. This
mutant carries missense mutation within 282-292 and fails to bind DNA.
5x109 bacterial cells induced for expression of the wild-type and mutant p53
proteins were lysed with 200 pi of 50 mM HEPES/10% sucrose solution
containing 0.5 mg/ml lysozyme. 1 pi of the antibody PAb421 (0.1 pg),
PAbl801 (0.1 pg), or buffer only was mixed and incubated with 1 pi of each
lysate containing the wild-type or mutant proteins as indicated at 0°C for 30
min, then mixed with 0.1 ng of the 3 2 P-labeled p53 binding sites containing
DNA BC for another 30 min. The protein-DNA complexes were resolved on
4% PAGE.
33
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DNA binding activity is detected in the presence (lanes 13-15) or the absence (lanes 13
and 15) of PAb421 for mP5C, which carries mutated conserved residues within 381-393.
Mutation at P3 and P2 Severely Diminishes the p53-TBP Interaction
Based on the work published by Prives and colleagues, we know that transcription
factor TBP can stimulate the sequence-specific DNA binding activity of p53 (Chen et al.,
1993). Lane and colleagues have shown that the sequence 370-382, which overlaps with
P3 (372-380), is responsible for maintaining the latent conformation for DNA binding.
Competition by peptide 370-382 of p53 can deform this conformation and activate p53
for DNA binding (Hupp et al., 1995). We therefore wished to determine whether the
DNA-binding stimulated by TBP is due to its binding at the C-terminus, including the P3
region, which would change the conformation of the DNA-binding domain of p53. The
ability of carboxyl-terminal missense mutants of p53 to interact with TBP when
compared with wild type was investigated by coimmunoprecipitation, in order to
determine which locus is critical for TBP-p53 interaction (Fig. 8). Wild type p53 and the
mutants mP5C, mP5U, mP3C, mP3U, mPl, mP2, mP4, and 1-380, described previously
were used for the assay. Among mPl, mP2, mP3C, mP3U, mP5C, and mP5U, with
mutations covering the carboxyl-terminal 343-393, mP3C (conserved residues within
372-380 mutated) shows a most serious decrease in TBP-binding activity (Fig. 8, upper
panel, lane 5). mP2, which carries the mutated sequence adjacent to that of mP3C, has a
similar decrease to P3 in this activity (lane 4). In contrast, mutants mP4, mPl, mP3U,
mP5C, mP5U, and 1-380 have binding capacities similar to wild type (compare lanes 2,
3, and 6-9 with 1). A consistent result was also obtained from coprecipitation using the
34
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£ o p U J D o
rv ^ r—i cn m m m m o o
f 1 11 1 11 12
1 2 3 4 5 6 7 89
Fig. 8. Mutation of P3 (372-380) or P2 (357-370) severely diminishes the TBP-
p53 interaction. The interactions of TBP with p53 and the mutants were
examined by coimmunoprecipitation. The p53 protein and its mutants were
expressed in bacteria and metabolically labeled with L-[3 5 S]methionine and L-
[3 5 S]cysteine. The GST-TBP chimeric protein was expressed in bacteria without
labeling. The bacteria were aliquoted, pelleted, and frozen at -80°C for the
experiments in the upper and lower panels. The protein expression was proved
and the protein quantities were examined in the lower panel before the
experiment in the upper panel was conducted. 5xl08 of the frozen cells were
lysed with 100 pi of PBS containing 0.25 mg/ml lysozyme. 25 pi of the lysates
containing the proteins to be tested were combined with 0.3 pg of the antibody
DO-1 and precipitated with PAA. Very similar amounts of all the proteins were
expressed in the same number of bacteria. Thus the same number of cells was
used in each precipitation in upper panel. Copricipitation of wild-typw p53 and
the mutants with TBP was conducted in the upper panel. The GS-4B bound TBP
chimeric proteins prepared from 25 pi of lysate were mixed with 25 pi of lysates
containing the wild-type or mutant p53 proteins as indicated. The unbound
proteins and impurities were removed by rinsing the GS-4B beads with EBC.
The proteins precipitated were resolved on 8% SDS-PAGE.
35
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Fig. 9. Comparison of the TBP-binding capacities between the intact C-
terminus of p53 and the truncated forms by coprecipitation. GST chimeric
proteins containing the full length of p53, the C-terminus of p53 293-393
and its truncated forms 293-383, 293-368, 325-393, 347-393, 352-393, and
357-393 were expressed in bacteria and then aliquoted, pelleted, and frozen
at -80°C. The cells were lysed with PBS containing 0.25 mg/ml lysozyme
and the proteins were precipitated with GS-4B from the lysates from 250 pi
of cells and resolved on 10% SDS-PAGE. Quantities of the proteins were
compared after Coomassie brilliant blue R-250 staining, and the adjusted
cells numbers from different samples were used to provide same amount of
the proteins for TBP binding later. The human TBP protein was expressed
from recombinant baculovirus in Sf9 cells. Cells were infecteded with the
TBP virus, metabolically labeled with L-[3 5 S]methionine and L-
[35S]cysteine 40 hour postinfection, and lysed with EBC containing 0.5%
NP40 in the ratio of 1.5xl06 cells/ml. The GS-4B bound proteins were
mixed with 300 pi of the TBP containing lyssate. The amount of the TBP
protein in lane 1 was 33% of the input in the other lanes. The unbound TBP
proteins were removed by rinsing the GS-4B beads with EBC. The bound
proteins on the beads were resolved on 10% PAGE.
36
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N- or/and C-terminal deletion mutants 293-393, 293-383, 293-368, 325-393, 347-393,
352-393, and 357-393 (Fig. 9). 293-383 (lane 3), bearing an intact P3, maintains a
comparable affinity for TBP when compared with 293-393 (lane 2), carrying an intact C-
terminal TBP-binding domain (317-393) (Horikoshi et al., 1993). However, the mutant
293-368 with deletion of P3 (372-380) plus partial deletion of P2 (357-370) loses TBP
binding activity (lane 5). Minimal binding by TBP could be detected even when the target
p53 sequence was as short as 357-393 (lane 9).
A Slight or Negligible Difference in Transactivity Is Detected between the Carboxyl-
Terminal Mutants and Wild-Type p53
To further demonstrate the contribution of residues 372-380 and 381-393 in the
regulation of transcriptional transactivity of p53 in vivo, a transient transfection-
transcription assay was undertaken by cotransfecting the POSTCAT2 reporter (kindly
provided by Dr. James Ou /Dr. Phillip H. Koeffler) with expression plasmids carrying
wild-type p53 and mutant cDNAs, encoding mP3C, mP5C, mP5U, 1-380, 1-366, 1-351,
and 1-342, into the p53-defective H358 non-small cell lung carcinoma cell line (from
ATCC). The CAT (chloramphenicol acetyl transferase) gene in POSTCAT2 is driven by
the Herpes simplex virus minimal thymidine kinase (tk) promoter preceded by 2 repeats
of the high affinity p53 binding site (Chumakov et al., 1993). Expression of wild type and
mutant p53 cDNAs is transcriptionally controlled by the mouse metallothionein-I gene
promoter (MT). No difference in CAT activity was detected between cells transfected
with wild-type cDNA (Fig. 10, lanes 2 and 11) or with cDNAs encoding mP5C (lane 6),
mP5U (lane 7), 1-380 (lanes 3 and 4), or mP3C (lane 8). There was slight increase in
37
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Fig. 10. The transient transfection-transcription assay to evaluate the effects of mutation
of P5 (381-393) and P3 (372-380) on the transcriptional activation function of p53. 2.5
pg of the POSTCAT2 reporter combined with 2.5 pg of the wild-type or mutant
expression plasmids as indicated were introduced into H358 non-small cell lung
carcinoma cells (p53-/-) by electroporation. The structures of the reporter and the most of
the mutants are introduced in the text and Fig. 2. mPl is a mutant which fails to form an
oligomer and to stimulate transcription. The transfected cells were grown in the presence
of 50 pM of ZnCl2, harvested 60 hours posttransfection, and lysed by freeze-thaw cycles.
Total protein concentrations of the lysates were measured with Bio-Rad protein assay dye
reagent. Endogenous acetyl transferase was heat-inactivated at 65°C for 10 min. The
same amount of total proteins in each reaction was mixed with 0.25 pCi of D-threo-
[dichloroacetyl-l-I4C] chloramphenicol and 1 pi of 40 mM acetyl coenzyme A in a total
volume of 100 pi, and incubated for at 37°C for 1 hr. The resulting products and the
unreacted substrates were separated from reaction mixtures by extraction with acetyl
acetate. The extracts were dried, resuspended in a small volume of acetyl acetate, loaded
on a silica gel coated plate, and resolved by thin layer chromatography as described in
Materials and Methods.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
38
Cont. wtp53 1-380 1-380 1-366
m m
4 1 •
# m
• •
#
•4
• •
m
2 3 4 5 1
mP5C mP5U mP3C mP2 mPl wtp53
d j j j l d
jiik jife
PP lP PP 41
•
• • • •
« • # *
'■v
• •
6 7 8 9 10 11
Fig. 10. The transient transfection-transcription assay to evaluate the
effects of mutation of P5 (381-393) and P3 (372-380) on the
transcriptional activation function of p53
39
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CAT activity from the cells transfected with the 1-366 (lane 5) and mP2 (lane 9) cDNAs,
when compared with the wild type.
Discussion
P5 in p53, Like in RB, Is Required for Maintenance of the Conformation in Which
the Protein Can Be Recognized and Phosphorylated by Cyclin A/Cdk2
The failure of mP5 and 1-380 to be phosphorylated by cyclin A/Cdk2 is possibly due
to loss of either the primary sequence or the conformation recognized by the kinase. The
latter possibility is supported by the evidence that when deletion is extended to residue
367, the resultant mutant (1-366) can be phosphorylated again. The explanation of this
observation is that residues 381-393 place residues 367-380 in a position such that they
are unable to inhibit phosphorylation. Loss of 381-393 allows 367-380 to assume the
“inhibition” conformation, which masks Ser3i5, and therefore prevents it from being
phosphorylated.
P5 Participates in the Sequence-Specific DNA-Binding Activating Process Triggered
by PAb421
The failure of mP5 and 1-380 (P5 deleted) to be activated for DNA binding by
PAb421 is apparently caused by inability of the molecules to adopt a coordinated
conformational change following the antibody interaction. This observation implies that
modification by PAb421 can not activate p53 for DNA binding unless the sequence 381-
393 joins other residues to establish a new conformation, which favors the protein-DNA
interaction. The explanation for the other observation, that the sequence 381-393 is not
required as soon as a further deletion reaches to residue 367, is that deletion of the partial
40
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of negative regulatory motif, 367-380, negates the DNA-binding inhibition. From these
data, a conclusion can be drawn: the sequence 381-393 functions as a counterpart to the
negative regulatory motif, in the regulation of the DNA binding activating process
triggered by PAb421 .
The conformation maintained by 381-393 is clearly required for both phosphorylation
by Cdk2 and sequence-specific DNA binding. The identity of sequence, which joins in
the conformation maintenance by interacting with 381-393, remains unclear.
Theoretically, independent mutation of each partner should affect the maintenance of
conformation that depends on their interaction. None of the mutants, with mutations in
343-351, 357-370, and 372-375, affected the phosphorylation by Cdk2. Thus, it is
unlikely that the sequence, which interacts with 381-393, is located within 343-375. We
therefore assume that the sequence, which interacts with 381-393 either intermolecularly
or intramolecularly, is located upstream of residue 343.
In summary, residues 381-393, in the inactive form of p53, contribute to maintenance
of the conformation required for phosphorylation by Cdk2, and they also, in the PAb421-
triggered activating process, participate in the creation of a new conformation that allows
p53 to bind the consensus DNA element.
P3 plus P2 Controls Both p53-DNA and p53-TBP Interactions
Consistent with the publication by Lane and colleagues, the sequence covered by P3
(372-380) plus P2 (357-370) maintains p53 in a latent state for DNA binding. Mutation at
P3 and P2 activates the p53-DNA interaction. In addition, we have found that the same
sequence antagonistically controls the p53-TBP interaction.
41
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There are two explanations for the sever decrease in the interaction between p53 and
TBP by the mutation of P3, despite the presence of an intact N-terminal domain 1) the
mutation has changed the conformation of the whole p53 molecule, which results in
masking of the N-terminal domain, or 2) the N-terminal domain in full length p53 is not
able to bind TBP without participation of other TFIID subunits.
The fact that minimal TBP binding by peptide 357-393 can be detected indicates that
P3 and P2 directly interact with TBP, and suggests that the activation of p53-DNA
binding by TBP may involve this interaction which disrupts the interaction of P3 and P2
with other residues. However, based on the results of our experiments, it is unclear
whether the interaction between TBP and the N-terminal activation domain of p53 is
required for the activation. To clarify this point, the activation domain-truncated p53
should be tested for DNA binding in the presence of TBP, to determine whether TBP can
activate the binding solely though interaction with the C-terminus of p53.
Mutation of P3 impairs the C-terminal domain, and therefore prevents p53 from
interacting with TBP in vitro. However, P3 missense and deletion mutants exhibit a
similar or slightly higher transactivity when compared with wild-type p53. This
observation implies that binding to TBP by the C-terminal domain is not required for the
transcriptional activation function of p53 once the protein is activated for DNA binding.
Possible Reasons for the Lack of Detectable Effect of P5 Mutation or Deletion on the
Transcriptional Activation Function of p53
Indistinguishable transactivation activity by mP5 and 1-380 when compared with
wild-type p53 may be due to several reasons, including: 1) Other pathways exist for in
vivo activation of p53 for DNA binding besides phosphorylation on Ser3is, such as
42
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phosphorylation by PKC on Ser37 8, and/or protein-protein interactions such as the TFIID-
p53 interaction; 2) In vivo, Ser3is in these mutants is still an viable substrate of Cdks; and
3) Different consensus binding sites, “BC” and “High Affinity p53 Binding Site”, were
used for the in vitro DNA-binding assay and transient transcription assay. Recent studies
have shown that phosphorylation by Cdks has a variable effect on the affinity of p53 to
different consensus binding sites. Phosphorylation increases the affinity of p53 binding to
the consensus sequence in the p21 gene regulatory element by more than 40 times, but
has no effect on binding to RGC (Wang and Prives, 1995). Considering that different
conformations are required by different enhancer-p53 interactions, the mutants which
failed to bind the sequence used in the gel shift assay might still be able to bind a
different sequence used in the transcription assay.
A Possible Reason for a Similar Transactivity Exerted by Both Wild-Type p53 and
DNA-Binding Constitutively Active Mutants
Transcriptional transactivities of both wild-type p53 and the mutants which are
constitutively active for DNA binding are similar to each other under our experimental
conditions. Based on the observation described very recently by Lozano and colleagues,
that wild-type p53 neither binds responsive elements nor transactivates target genes in the
GO phase of the cell cycle compared with the G1 phase (Deffie et al., 1995), an
explanation for our result is that modification of p53 occurs as soon as cells enter the cell
cycle, and this can highly activate wild-type p53 to a level comparable with the DNA-
binding constitutively active mutants mP3C, mP2, and 1-366. Our experiment is
performed using actively dividing cells. This may disallow detection of a difference in
43
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transactivity. To resolve this problem, quiescent transfected cells should be analyzed for
comparison of transactivities of wild-type p53 and the mutants.
Conclusion
1. P5 (381-393) is at least part of the sequence that determines the conformation of
p53 that allows access to and phosphorylation of Ser3is by cyclin A/Cdk2 in vitro.
2. P5 (381-393) is required for creation of the new conformation of p53 that favors its
interaction with consensus DNA in the activating process triggered by binding of
antibody PAb421.
3. P3 (372-380) and P2 (357-370) form a functional motif that antagonistically
controls the p53-DNA and p53-TBP interactions.
Materials and Methods
Plasmids and Recombinant Baculoviruses
To create bacterium expression p53 plasmid pETwtp53, the wild-type human p53
cDNA in the pBSH 19(A) plasmid (a gift from Dr. Miller) was digested with BamH I and
partially digested with Nco I, and cloned into the pET8 C vector. Using pETwtp53 as a
back bone, the missense mutation mutants were constructed by replacing the wild-type
sequences for the corresponding sequences which carry the mutations created by RCR
directed mutagenesis. pETp53mP5C was constructed with the secondary PCR product
which was produced using one regular primer at 3’-end and one extended primer at 5’-
end. The extended primer was created by primary PCR in which two short and partially
44
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complementary DNA fragments were annealed and extended by Taq DNA polymerase.
The resulting longer DNA containing the sequence encoding all the missense mutated
amino acid residues was used as the 5’-end primer for the secondary PCR. pETp53mP3C’
and pETp53mP3C were constructed using the one time PCR fragments. The sequence
encoding first four mutated amino acid residues 372-375 were introduced into the wild-
type cDNA with Stu I and Acc I sites to create pETp53mP3C’. Continuously, the
sequence encoding 2 mutated amino acid residues 378 and 380 were introduced into
mP3C’ cDNA with Acc I and BamH I to create pETp53mP3C. pETp53mPl and
pETp53mP4 were constructed as follows: four primers A, B, C and D were used and two
times of PCR were performed. The first PCR created two fragments 5’A— B 3’ and 5’C—
D 3’. Among the four primers, B and C carried missense mutations at their 5’ ends
therefore only 3’ ends of the primers completely matched to the template DNA. The
overlapped parts of these two primers contained part or all of the mutated codons and
were 100% complementary to each other rather than to the template. This design provided
the possibility for annealing of A— B and C--D in second PCR. A and D were regular
primers located respectively at 5 -end and 3’-end of the two primary PCR products in
respect to the open reading frame of p53. First five cycles for second PCR were carried
out with one tenth amount of the purified A— B and C— D from first PCR without primers
thus the two fragments could be annealed and extended to become A— B/C--D with a
molecular size of A— B plus C— D. Then primers A and D were added and 25 more cycles
were continued to create abundant A--B/C— D fragments. The secondary PCR products
were digested with proper restriction endonucleases, purified, and used to replace the
45
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corresponding wild-type sequences. pETp53mP5U, pETp53mP3U, and pETp53mP2
were constructed by Dr. Xu-Xian Zhang. The truncated p53 mutant plasmids pETp53/l-
366, pETp53/l-351, and pETp53/l-342 were constructed with Bsu36 I and BamH I
digested pETwtp53 and the corresponding PCR fragments. pGEX-TBP was constructed
by graduate student Ling Li, with pGEX2T (Pharmacia) as the expression vector and
plasmid GPP-63 (a gift from Dr. Peterson) as the source of human TBP cDNA. GST
fusion p53 truncated mutants pGEXp53/347-393, pGEXp53/352-393, and
pGEXp53/357-393 used for coprecipitation with TBP were constructed by ligation of the
BamH I and EcoR I digested pGEX2T vector and the same enzymes digested PCR
fragments (the enzyme sites were artificially designed in the PCR primers). The truncated
mutants pGEXp53/293-393, pGEXp53/293-383, pGEXp53/293-368, and pGEXp53/325-
393 used for the same experiment as above were constructed by Dr. Chen Li by the same
method. pGEXp53 was constructed by Dr. Takashi Koyama by cloning the 5’-end
reformed p53 cDNA with BamH I into pGEX2T. To construct eukaryotic gene
expression plasmids carrying wild-type p53 and the mutants for the transient transfection-
transcription assay, p53 and the mutant cDNAs from the reformed pETwtp53 and the
mutants were (with BamH I sites at both 5’ and 3’ of the cDNAs) digested with BamH I
and cloned into the corresponding enzymes digested MTneo vector to create MTwtp53
and the mutants MTp53mP5C, MTp53mP5U, MTp53mP3C, MTp53mP2, MTp53mPl,
MTp53/l-380, and MTp53/l-366. The reporter POSTCAT2 was provided by Dr. James
Ou/Dr. Phillip H. Koeffler (Chumakov et al., 1993).
46
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The human TBP recombinant baculovirus was constructed by in vivo recombination
between the pACYMl-hTBP plasmid and the Bsu36 I digested Bakpack 6 baculoviral
DNA in insect Sf9 cells. The method is described in detail in Materials and Methods of
Chapter 3. The Cyclin A and Cdk2 recombinant baculoviruses were gifts from Dr. David
Morgan.
Bacterial Strains and Insect and Mammalian Cells
E. coli strain BL21 was used as the host for protein expression from plasmids with
pET8 C backbone. E. coli strain DH5a was used as the host for protein expression from
plasmids with pGEX2T backbone. Both bacteria were grown in LB medium at 37°C
unless illustrated.
Insect Spodoptera frugiperda (Sf9) cells used as the host cells for baculoviral
infection and protein expression were purchased from ATCC. The cells were grown in
TNM-FH medium (GIBCO BRL) containing 10% fetal bovine serum (FBS), 50 units/ml
penicillin G, and 50 pg/ml streptomycin at 26°C.
Non-small cell lung carcinoma line H358 (p53-/-) used for the transient transfection-
transcription assay was provided by Dr. John Minna. The cells were grown in R P M I1640
medium (GIBCO BRL) containing 10% FBS, 100 units/ml penicillin G, and 100 pg/ml
streptomycin at 37°C in the presence of 5% C02.
In Vitro Phosphorylation
The procedures for preparation of crude cyclin A/Cdk2 holoenzyme mainly follow the
method as described (Driscoll et al., 1995).
47
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1 x 107 actively growing Sf9 cells in a T150 tissue culture flask were coinfected with
cyclin A and cdk2 recombinant baculoviruses for 1 hr at 26°C. The fresh medium was
added after the infectous medium was vacuumed out. The cells after 48 hour infection
were knocked off from surface of the flask, transferred with the medium to a centrifuge
tube, and pelleted in an EEC medical centrifuge at grade 3 for 3 min at room temperature.
The cells were rinsed one time with phosphate-buffered saline (PBS) (140 mM NaCl, 2.7
mM KC1, 10 mM Na2HPC> 4, 1.8 mM KH2PO4), pH 7.4, and lysed at 4°C in 500 pi of
kinase reaction buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCh, and 10 mM 1 3 -
mercaptoethanol; proteinase inhibitors: 5 pg/ml aprotinin, 5pg/ml leupeptins, 0.1 mM
PMSF; and phosphatase inhibitors: 0.1 mM NaF, 10 mM 8 -glycerophosphate, 0.1 mM
sodium orthovanadate) supplemented with 150 mM NaCl and 0.5% NP40.
Bacterial cells carrying the plasmids encoding wild-type p53 and the mutants were
induced with 1 mM of IPTG for protein expression at room temperature for 3-5 hrs. The
cells were harvested by centrifugation. The pellets were suspended in 100 pi of PBS, pH
8.4, containing 0.25 mg/ml lysozyme, proteinase inhibitors (3 pg/ml aprotinin, 3 pg/ml
leupeptins, 3 pg/ml pepstatin A, 0.1 mM PMSF) and 100 pg/ml DNase I, and maintained
on ice for 10 min. The cells were lysed by freezing in dry ice/ethanol bath for 5 min and
thawing in 37°C water bath for 1 min. The treatment was repeated for 2-3 more times.
Cell debris was removed from the lysates by centrifugation at 14k rpm for 10 min at 4°C.
The proteins from the lysates were bound by anti-human p53 monoclonal antibody
PAbl22 or DO-1, immobilized to protein A-agarose (PAA), and rinsed with the kinase
reaction buffer at 4°C for three times.
48
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25 |al of the crude kinase was mixed with the substrate-antibody complex
immobilized on PA A. Kinase reaction was carried out at 30°C for 1 hr by adding 10 pCi
of [y-32P]ATP (6,000 Ci/mmole, from NEN) adjusted with unlabeled ATP to a final
concentration of 25 pM. The proteins immobilized by antibodies on PAA after the kinase
reaction were rinsed with EBC buffer (20 mM Tris-HCl, pH 8.0, 125 mM NaCl, 0.5%
NP40, 2 mM EDTANa^, and phosphatase inhibitors: 20 mM NaF and 0.1 mM Na3V 0 4 )
and repelleted by centrifugation. Rinse-pellet was repeated for two more times. The
samples were resolved on SDS-PAGE. The results were read from X-ray film (Kodak)
exposed to the 1 0% acetic acid and 1 0 % ethanol fixed and heat-dehydrated gel.
The protocol above mainly follows that in the publication by Dr. Barbara Driscoll
(Driscoll et al., 1995).
The Gel Electrophoretic Mobility Shift Assay (EMSA)
Preparation of 32P-labeled double-stranded DNA. Two single strands of synthesized,
complementary and OPC column (Applied Biosystems) BC sequence
5’CCGGGCATGT/CCGGGCATGT/CCGGGCATGT3’ and 5’GGCCCGTACA/GGCC
CGTACA/GGCCCGTACA 3’ 2 pg/pl each are mixed with equal volume in 1/25 x
kinase buffer, denatured at 90°C for 2 min, transferred to a 65°C water bath for 3 min, and
annealed in a 65°C small water bath until the temperature decreased to 25°C or lower. 2
pi of the annealed DNA were labeled at 37°C for 1 hr in 3 pi of blunt end mix buffer (0.2
M Tris-HCl at pH 9.5, 1 mM EDTA, and 10 mM spermidine), 5 pi of lOx kinase buffer,
24 pi of ddH20, 1 5 pi (150 pCi) [y-32P]ATP, and 1 pi of T4 polynucleotide kinase
(NEB). The reaction was terminated by adding 2 pi of 0.5 M EDTANa2, pH 8.0. The
49
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labeled probe was separated from the free [y-32P]ATP by passing through a G50 column
(=0.5 ml of beads) and eluted with 10 mM Tris-HCl and 0.1 mM EDTA, pH 7.4. Eluting
fractions were collected in 1 0 0 p 1/tube. 1 pi from each tube was counted for cpm in a
scintillation counter (Wallac/Pharmacia, 1209 Backbeta). Fractions with high cpm were
pooled and stored at -20°C.
Cells induced for expression of wild-type p53 or the mutant as described in the
previous section were pelleted, frozen quickly in an eppendorf tube in liquid nitrogen in
50 mM HEPES, pH 8 .6 , and 10% sucrose in the ratio of 100 pl/10 ml of bacteria, and
stored at -80°C. The frozen bacteria were thawed on ice. Extraction buffer was added to
the bacteria to final concentrations of 0.25 M KC1, 2 mM of DTT, 0.5 mg/ml lysozyme,
0.2 mM PMSF, 5 pg/ml pepstatin A, 5 pg/ml luepeptins, and 5 pg/ml aprotinin. The
bacteria were rocked at 4°C for 1 hr. The cell lysate was centrifuged at 14K rpm for 10
min at 4°C and the supernatant was transferred to a new eppendorf tube.
The crude extracts were mixed with 1 pi (0.1 pg) of anti-p53 antibody and 3 pi of
DNA binding buffer (25 mM HEPES at pH 7.6, 50 mM KC1, 1 mM DTT, 1 mg/ml BSA,
and 0.1% Triton-X 100) and incubated on ice for 30 min. 15 pi of reaction solution ( 6 pi
of 5x DNA binding buffer, 4 pi of 15% ficoll, 6 pi of 20 mg/ml bovine serum albumin
(BSA), 1 pi of 100 pg/ml sonicated salmon sperm DNA with MW of 500 bp, and 32P-
labeled BC with a radioactive intensity of 104 cpm) were added to the mixture.
Incubation was undertaken at room temperature for 20 min. 3pl of termination solution
(15% ficoll, 0.5% bromophenol blue and 50 mM EDTA) were added to each reaction.
50
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PAGE used for electrophoresis was made as the following recipe: 18.4 ml of ddH20,
1.5 ml of 5X TBE, 2.25 ml of 40% acrylamide, 220 pi of 10% Triton X-100, 150 pi of
10% ammonium persulfate and 25 pi of TEMED. A total volume of the mixture was
adjusted to 22.5 ml. The gel was prerun in 1/3 X TBE and 0.1% triton X-100 at 4°C under
8 mA/plate for 15-30 min. The samples were loaded with power on. The gel was run until
bromophenol blue reached the middle of the vertical length. The whole procedure for
electrophoresis above was carried out at 4°C. The gel was dried on a gel drier at 80°C for
30 min. Result was visualized by exposure of the gel to X-ray film.
Immunoprecipitation
1 ml of growing bacteria in logarithmic phase carrying the expression plasmids was
pelleted in an eppendorf tube by centrifugation and resuspended in 200 pi of fresh LB
with 3 mM IPTG. 100 pCi of Expre35S3 5 S containing L-[35S]methionine and L-
[35S]cysteine (NEN) were added to the cells and incubation was undertaken at room
temperature for 3-5 hrs. The cells were harvested by centrifugation in a microfuge. The
pelleted cells can be either used freshly or frozen at -80°C for later use. The pellets were
suspended and lysed as described in the section of In Vitro Phosphorylation. Cell debris
was removed from the lysates by centrifugation at 14k rpm for 10 min at 4°C. The
supernatant was transferred into a new eppendorf tube.
For testing protein-protein interaction, two supernatants (25pl) each containing one of
two studied proteins were mixed in an eppendorf tube and adjusted to 300 pi with EBC
lysis buffer (20 mM Tris-HCl, pH 8.0, 125 mM NaCl, 0.5% NP40, 2 mM EDTANa2,
phosphatase inhibitors: 20 mM NaF and 0.1 mM Na3V 0 4 , and proteinase inhibitors: 1
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pg/ml aprotinin, 1 pg/ml leupeptins, 1 pg/ml pepstatin A, 0.1 mM PMSF). The mixture
was rocked at 4°C for 1 hr. 0.2-0.3 pg of antibody against either one of the two tested
proteins was added. Rocking was continued for 1 more hr. The mixture was centrifuge
briefly and 15 pi of PAA larry, EBC:PAA =1:1, saturated with BSA was added. Rocking
was continued for 1 more hr. The protein-protein-antibody complexes bound on beads
were rinsed with 1ml of cold EBC (20 mM Tris-HCl, pH 8.0, 125 mM NaCl, 0.5% NP40,
2 mM EDTANa2), pelleted in a microfuge at 12k rpm briefly (=5 seconds). EBC was
removed by vacuum. Rinse-pellet-vacuum was repeated for 2 more times.
For testing GST fusion protein-nonfusion protein interaction, 25pl of the supernatant
containing GST fusion proteins were diluted to 300 pi with the EBC lysis buffer, mixed
with 10 pi of glutathione conjugated sepharose 4B (GS-4B) larry, and rocked at 4°C for 1
hr. The proteins bound on beads were rinsed with 1 ml of 4°C EBC for 2 times and
resuspended in 300 pi of EBC lysis solution. The solution containing purified GS-4B
bound GST fusion proteins was mixed with the supernatant containing 25 pi of non
fusion proteins in 300 pi of total adjusted volume with EBC lysis solution and rocked at
4°C for 1 hr. The complexed proteins bound to GS-4B beads were rinsed 3 times with
4°C EBC.
The rinsed beads were suspended with 20 pi of 2x loading buffer and heated at 8 6 °C
for 10 min. The heated samples were briefly centrifuged and the supernatants were loaded
to wells of SDS-PAGE. The gel was run with a low voltage (=60V) overnight or with a
high voltage (=200V) for several hours until the bromophenol blue dye reached the
bottom. The gel was stained in 0.25% Coomassie brilliant blue R-250, 45% methanol,
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45% water, and 10% acetic acid for 15-30 min and destained with 10% acetic acid and
10% ethanol in water. The gel was dried at 80°C. One sheet of X-ray film was loaded on
the dehydrated gel and developed at proper time.
The recipes for making SDS-PAGE, 2x gel loading buffer, gel running buffer, and
staining solution were adopted from Molecular Cloning by Sambrook, Fritsch, and
Maniatis.
The Transient Transfection-Transcription Assay
Plasmid DNAs for the assay were prepared by chromatography using anion-exchange
resin-filled column GLAGEN-tip (from QIAGEN), and stored as 70% ethanol stocks until
use. The DNAs were pelleted by centrifugation, rinsed with 70% ethanol, air dried in a
tissue culture hood, and suspended in 10 mM Tris-HCl and Im M EDTANa2, pH 8.0.
H358 cells were grown to 80% confluence. Medium was changed several hours before
transfection. The cells were rinsed with PBS, pH 7.4, and released from surface of the
flask with 0.05% trypsin and 7 mM EDTA solution. Digestion was blocked with 10
volumes of the RPMI 1640 medium containing 10% FBS. The cells were pelleted in an
IEC medical centrifuge at grade 3 for 3 min. The cells were rinsed with RPMI 1640
medium containing 6 mM glucose, repelleted, and suspended to a density of 3 x 106 per
0.9 ml of the same medium. 2.5 pg of the POSTCAT2, and 2.5 pg of wild-type p53
plasmid, MTwtp53, or equal molar number of its mutants were used for each transfection.
The difference of DNA amount between p53 and the mutants (p53 and the mutants had
different molecular weight) was filled up with blank plasmid MTneo. The mixed DNAs
were diluted to a final volume of 100 pi with 10 mM Tris-HCl and 1 mM EDTANa2, pH
53
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8.0. 0.9 ml of suspended cells was transferred to each chamber (GIBCO BRL). The DNA
mixture was added to the chamber and mixed with the cells by turning the chamber up
and down for several times. An electric shock was triggered to each chamber in a BRL
Cell-Porator under the conditions of 220 volts, 1180 Farad, and low The cells were
diluted 10 min after electroporation with RPMI 1640 medium with FBS and antibiotics,
and dispensed into a 100 mm cell culture dish. The medium was changed the next day.
The transfected cells 48-60 hours postelectroporation were washed 5 times with PBS,
pH 7.4. 1 ml of TEN (40 mM Tris-HCl at pH 7.5, 10 mM EDTA, 150 mM NaCl) was
added onto the cells. The cells were scraped from each dish, transferred into an eppendorf
tube and pelleted by centrifugation at 6,000 rpm for 3 min. Supernatant was removed and
replaced with 160 pi of 250 mM Tris-HCl, pH 8.0. The pellets were resuspended and
subjected to three freeze-thaw lysing cycles on dry ice and in 37°C water bath.
Supernatants were recovered by centrifugation at 14k rpm for 10 min at 4°C. A small
aliquot of the supernatant from each sample w as diluted and mixed with Bio-Rad reagent
to a final 1 ml of volume for total protein concentration measurement in a
spectrophotometer with the 595 nm wave length. The rest of supernatant was heated at
60°C for 10 min to inactivate endogenous acetylase, and centrifuged at 4°C to eliminate
the denatured proteins. The crude protein extracts recovered were either frozen at -80°C
or freshly used for the chloramphenicol acetyl transferase (CAT) assay.
Based on the measured total protein concentrations above, same amount of total
proteins from the crude cell extracts was taken, adjusted to 80 pi with 250 mM Tris-HCl
at pH 8.0, and mixed with 0.25 pCi/5 pi of D-threo-[dichloroacetyl-l-
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14C]chloramphenicol (Amersham) and 1 pi of 40 mM acetyl coenzyme A (Sigma) diluted
in 14 pi of 250 mM Tris-HCl buffer, pH 8.0. The reaction was undertaken at 37°C for 1
hr. Each reaction mixture was extracted with 300 pi of acetyl acetate for 2 times. Acetyl
acetate was separated from aquatic phase by centrifugation at 14K for 3 min, and
transferred to a new eppendorf tube. Total 600 pi of the extracts from each reaction were
dried in a speed vacuum condenser. The pellets were resuspended in 15 pi of acetyl
acetate and loaded dropwise on a silicon thin layer chromatography plate (Whatman). The
plate was subjected to chromatography in chloroform/methanol (95:5) in a tightly closed
glass tank until the front of the solvent reached 2 cm from the top. The plate was air
dried, wrapped with seal wrap, and exposed to X-ray film. The enzyme activities from
different samples were compared by vision or by cutting the spots of products from the
plate and counting cpm in a scintillation counter.
The procedures for the CAT reaction above were mainly referenced from the chapter
of Identification of Regulatory Elements of Cloned Genes with Functional Assays by
Nadia Rosenthal in Vol. 152 of Methods in Enzymology.
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CHAPTER 3: DISCOVERY OF BINDING OF D-TYPE CYCLINS TO p53 AND
FUNCTIONAL STUDY OF THE INTERACTION
Summary
In the study of the function of P5 described in Chapter 1, our laboratory found that P5
is a motif needed to maintain the conformation required for RB phosphorylation by Cdk2.
We were wondering then which Cdk(s) can phosphorylate p53 and whether
phosphorylation requires the presence of P5 sequence. We tested different Cdks for their
kinase activities on human p53. A direct interaction of p53, in particular the truncated
form, with baculoviral recombinant human cyclins D l, D2, and was observed even
though no phosphorylation of p53 by cyclin D/Cdk4 was detected. By
coimmunoprecipitation, two independent cyclin D binding domains were defined within
residues 100-160 and 237-292, covering three (II, IV, and V) of the five p53 domains of
high homology among different species (Soussi et al., 1990). The cyclin D-binding
domains overlap with the sequence-specific DNA-binding domain (100-292) where a
majority of p53 point mutations occur in human cancers.
We have also determined the sequence-specific DNA binding domain in p53 using
EMSA. When DNA- and cyclin D-binding characteristics of p53 were compared, it was
found that deletion of the carboxyl terminal 40 or more residues completely activates both
bindings. However, binding of the monoclonal antibody PAb421 to P3 (372-380) or
mutation of this sequence fully activates DNA binding but only negligibly activates the
cyclin D binding.
To understand the significance of the direct interaction between cyclin D and p53, we
have pursued two avenues of investigation. First, effect of binding of the truncated p53,
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1-351, by cyclin D2 on 1-351-DNA complex formation was evaluated by EMSA. The
result showed that 1-351/cyclin D2 complex apparently does not form a tripartite
complex with the p53 responsive elements. Second, exclusion of binding of either 1-351
or RB by the other, when bound by cyclin D3, was examined using
coimmunoprecipitation. RB was not detected in the cyclin D -1-351 complex precipitated
with anti-p53 antibody; and 1-351 was observed at a very low level in the cyclin D-RB
complex precipitated with anti-RB serum. A well-known function of tumor suppressor
p53 is the transcriptional activation of p21/Wafl/Cipl in response to DNA damage. This
gene codes for a potent inhibitor of cyclin-dependent kinases, which can prevent key cell
cycle regulators such as RB from being phosphorylated and whereby induce G1 arrest.
The results from our experiments imply a new aspect of the functional antagonization
between p53 and D-type cyclins.
Introduction
Cell Cycle Control by Cyclins and Cyclin-Dependent Kinases
Cyclin-dependent kinases (Cdks) control the transitions between successive phases of
the cell cycle in all eukaryotic cells (reviewed by Nurse, 1990, Mailer, 1991, Norbury and
Nurse, 1992, Reed, 1992, and Nurse, 1994). In order to be active, the catalytic subunit
(Cdk) needs to assemble into a complex with a cyclin which functions as a regulatory
subunit (Connell-Crowley et al., 1993) and maybe also as an adaptor for Cdks to the
substrates (Kato et al., 1993). In addition, activation of Cdks requires the displacement of
Cdk inhibitors (CKIs) including pl5, p l6 , p21, p27 and p57 (Kato et al., 1994a; Polyak et
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al., 1994a and 1994b; Toyoshima et al., 1994; Serrano et al., 1993; reviewed by Elledge
and Harper, 1994; Lee et al., 1995; Matsuoka et al., 1995), phosphorylation on threonine
by cyclin-dependent kinase activating kinase (CAK) (Fisher and Morgan, 1994; Matsuoka
et al., 1994; Kato et al., 1994b), and dephosphorylation on Thrl4 and Tyrl5 by CDC25
family members (Dunphy and Kumagai, 1991; Gautier et al., 1991; Struasfeld et al, 1991;
reviewed by Dunphy, 1994). Phosphorylation by activated Cdks of their target proteins
such as RB on serine/threonine residues drive cells into S phase.
The cell cycle of somatic cells consists of four phases G l, S, G2 and M. DNA
replication occurs during S phase and chromosome segregation occurs during M phase. S
and M phases are separated by gaps Gl (before S) and G2 (before M). Orderly cell cycle
progression demands that Cdks be precisely regulated, becoming active and inactive at
appropriate times. Transcriptional regulation and proteolytic degradation of cyclins are
two of the principle strategies adopted by cell cycle machinery to switch on and off the
Cdk activities (Draetta et al., 1989; Glotzer et al., 1991; Lew, et al., 1991; Koff, et al.,
1991; Murray, 1995; Irniger et al., 1995; King et al., 1995; Tungendreich et al., 1995).
Based on the specific cell cycle interval when they accumulate and function, mammalian
cyclins are divided into G l (cyclin D and cyclin E) (Matsushime et al., 1991; Lew et al.,
1991; Koff et al., 1991; Dulic et al., 1992), S (cyclin A) (Girard, 1991) and G2/M (cyclin
A and B) (Pines and Hunter, 1990; Roy et al., 1991) cyclins. Unlike yeast cells, where
cell cycle progression is controlled by a single Cdk molecule encoded by cdc2 (in
Schizoscicchciromyces pombe) or by CDC28 (in Saccharomyces cerevisiae), a series of
Cdks function to stimulate different cell cycle transitions associating with their specific
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cyclin partners in higher eukaryotes. Cyclin B forms a complex with Cdc2 to regulate
both mitotic entry and exit (Gautier et al., 1990; Nurse, 1990; Mailer, 1991). Cyclin A
associates with Cdk2 during the S phase and with Cdc2 during the G2 phase respectively
(Draetta et al., 1989; Giordano et al., 1989; Pines and Hunter, 1990 and 1991; Pagano et
al., 1992; Rosenblatt et al., 1992; Elledge, et al., 1992). Evidence suggests that cyclin A
may act to ensure the temporal relationship between S phase and mitosis (Girard, 1991;
Walker and Mailer, 1991; Pagano et al., 1992; Zindy et el., 1992). Cyclin E, like cyclin A,
associates with Cdk2 but in late G l and S, to control the Gl/S transition (Koff et al.,
1991; Dulic et al., 1992). D-type cyclins consist of three closely related G l cyclins D l,
D2 and D3. In insect cells, all three of them are able to activate Cdk4 and Cdk6 , but only
D2 and D3 can activate Cdk2 (Matsushime et al., 1992; Ewen et al., 1993; Meyerson and
Harlow, 1994). The primary function of D-type cyclins is to stimulate G l progression
(Matsushime et al., 1991; Baldin et al., 1993; Jiang et al., 1993; Quelle et al., 1993;
Resnitzky et al., 1994).
D-type Cyclins Stimulate Cell Cycle Progression in the G l Interval and Prevent
Cells from Differentiation
Unlike other cyclins, which are tied to the cell cycle clock, D-type cyclins act as an
integrator of environmental signals including mitogenic and antimitogenic stimuli that
determine whether the cell should enter or exit cell cycle (reviewed by Sherr, 1994). For
examples, the D-types cyclins in macrophages or T lymphocytes are induced by colony-
stimulating factor-1 (CSF-l/M-CSF-1) during G l and their continuous synthesis depends
on persistent growth factor stimulation (Matsushime et al., 1991; Ajchenbaum et al.,
1993); and D2, D3, and Cdk4 undergo degradation in myeloid cells induced for
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differentiation (Kato and Sherr, 1993) with granulocyte-colony stimulating factor (G-
CSF). One of the main targets of cyclin D2 and D3 is believed to be retinoblastoma
susceptibility gene product (RB) which is a key regulator of cell growth (Goodrich et al.,
1991), essential to mouse development (Lee et al., 1992; Jacks et al., 1992; Clarke et al.,
1992), and required to produce and maintain the terminal differentiated phenotype of
muscle cells (Gu et al., 1993). The mechanism for cyclin D/Cdk4 or Cdk6 in cooperation
with cyclin E/Cdk2 to promote the Gl progression is proposed to involve
phosphorylation and concomitant inactivation of RB for its Gl/S transition inhibition
function (Hinds et al., 1992; Kato et al., 1993; Ewen et al., 1993). Besides Gl
stimulation, overexpression of D2 and D3 prevent differentiation of myeloid cells in G-
CSF (Kato and Sherr, 1993). Cyclin D l, like D2 and D3, physically interacts with pRB,
but this doe not lead to its phosphorylation in cotransfected osteosarcoma SaoS2 cells.
Reversely, RB acts as a regulator ;o sequester cyclin D l possibly by their direct
interaction (Dowdy et al., 1993).
Molecular Genetics of D-Type Cyclins
Three cDNAs of mouse D-type cyclin genes Cyll, Cyl2, and Cyl3 have been isolated
using their messenger RNA-enriched macrophages induced with CSF-l/M-CSF-1 by
Matsushime and colleagues (Matsushime et al., 1991), and three human homologues have
been isolated with distinct strategies by Lew, Motokura, Withers, Xiong, and their
colleagues (Lew et al., 1991; Motokura et al, 1991; Withers et al, 1991; Xiong et al., 1991
and 1992). Human D-type cyclins D l, D2 and D3 are encoded by CCND1, CCND2 and
CCND3 genes on discrete genomic loci of chromosomal 1 Iq 13, 12pl3 and 6p21
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respectively (Motokura et al., 1991; Withers et al., 1992; Xiong et al., 1992b). The
calculated molecular weights of the three expressed proteins are 33.67, 33.05, and 32.48
kD. D-type cyclins are partially cell type specific, with most of cells expressing D2 and
D1 or D3 (Matsushime et al., 1991; Inaba et al., 1992).
Cyclin D l: a Proto-Oncogene
At the same time when human cyclin D l was identified and isolated as a cDNA
complementary to the yeast G1 deficiency (Xiong et al., 1991), it was also identified and
isolated as a putative oncogene (Motokura et al., 1991; Withers et al., 1991): in
parathyroid adenomas, human B-cell leukemia, and lymphomas. Over-expression of
cyclin D l (then named as PRAD1) in parathyroid adenoma was as a result of its gene
CCND1 (in llq l3 ) juxtaposed to the 5’ regulatory region of parathyroid hormone gene
(in llp l5 ) and stimulated by its promoter (Arnold et al., 1989; Rosenberg et al., 1991;
Motokura et al., 1991). A t(lI;14)(ql3;q32) translocation was found in human B-cell
leukemia and lymphomas. The breakpoints at chromosome 14q32 occur in the jointing
region of the immunoglobulin heavy-chain gene and the locus of breakpoints at
chromosome 11 is called bcl-1 (B-cell leukemia/ lymphoma 1) (Tsujimoto et al., 1985;
Rabbitts et al., 1988; Koduru et al., 1989; Meeker et al., 1989; Withers et al., 1991; Seto,
M. et al., 1992). The cyclin D l gene locus is also amplified and its mRNA is over
expressed in several types of human tumors (Lammie et al., 1991; Jiang et al., 1992;
Buckley et al., 1993; Tsuruta et al., 1993; for review, see Motokura and Arnold, 1993).
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p53 Functions to Antagonize D-Type Cyclins
G1 interval is the time when cells are preparing for DNA synthesis and D-type cyclins
are governing this progress. DNA damage caused by exposure to genotoxic reagents
before cells enter the S phase must be repaired, otherwise become fetal or cancer
promotive (Kastan et al., 1992; Lane, 1992, and references therein). The well-established
function of the tumor suppressor p53 is its acting as a checkpoint factor to induce G1
arrest in DNA damaged cells, providing enough time for DNA repair before they enter
next cycle. The p53-dependent G1 arrest is believed mainly through transcriptionally
activating the downstream effectors such as p21/WAFl/Cip-l of Cdk inhibitor (El-Deiry
et al., 1993; Gu et al., 1993; Harper et al., 1993; Xiong et al., 1993). The increased p21
proteins can form a complex with and inhibit G1 cyclin-Cdk complexes such as cyclin
D/Cdk4 from phosphorylating RB and possibly other substrates thereby to cause G1
arrest (Matsushime et al., 1992; Xiong et al., 1993; Ewen et al., 1993; Dowdy et al.,
1993).
Discovery of p53-Cyclin D Interaction
In addition to trying to find out whether P5 (residues 381-393) is responsible for
maintaining the conformation required for phosphorylation of p53 by Cdk2 in our
research, we also examined what else type of Cdks phosphorylates p53 (by Dr. Barbara
Driscoll) besides Cdk2. Surprisingly, we observed that p53, in particular the truncated
form, associated with all three human cyclin D family members D l, D2 and D3 even
though p53 could not be phosphorylated by the cyclin D/Cdk4 complexes. Further
investigation of p53-cyclin D interaction defined two cyclin D binding domains within
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the core region of p53. The conformation requirement for cyclin D-binding was compared
with that for its DNA-binding (Hupp et al., 1992, and data from our laboratory) and that
for replication inhibition (Cox et al., 1995). The relations between truncated p53-DNA
and truncated p53-cyclin D interactions and between cyclin D-RB and cyclin D- truncated
p53 complex formations were also explored.
Results
Human D-Type Cyclin Family Members Preferentially Interact with the Truncated
Forms of p53
The interactions between human p53 and three human D-type cyclins, D l, D2, and
D3, were demonstrated by coimmunoprecipitation. Insect Sf9 cells were infected with
p53 recombinant baculovirus, with cyclin D recombinant baculovirus, and with both. The
proteins in the infected cell lysates were precipitated with anti-p53 monoclonal antibody
DO-1, and with anti-D-type cyclin rat monoclonal antibodies Ab-1 to D l, Ab-1 to D2,
and Ab-1 to D3 respectively. All three family members cyclin D l, D2, and D3
preferentially interacted with the C-terminus truncated form of p53, 1-292 (Fig. 1, lanes
6 -8).
Upon knowing that the cyclin D binding is stimulated by deletion of the C-terminal
101 residues of p53, we tested how long the minimal sequence needs to be deleted in
order to optimally activate the p53-cyclin D interaction and whether deletion of the N-
terminus also affects this interaction. The same experiment was conducted as above using
recombinant baculoviruses carrying the p53, mP3C, mP5C, 1-366, 1-351, 1-342, and 6 6 -
393 cDNAs (Fig. 2). The result showed that deleting the C-terminal 42 residues was
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wtp53 1-292
D l D2 D3 D l D2 D3 D l D2 D3
-66
-45
-31
1 2 3 4 5 6 7 8 9 10 11
Fig. 1. Identification of the interactions of p53 and the deletion mutant 1-292
with human cyclin D l, D2, and D3 by coimmunoprecipitation. Insect Sf9
cells were infected with individual recombinant baculoviruses carrying the
p53, truncated p531-292, cyclin D l, D2, and D3 cDNA (lanes 1, 5, and 9-11)
coinfected with two recombinant baculoviruses carrying the p53 and cyclin
D l, D2, or D3 cDNAs (lanes 2-4), or coinfected with two recombinant
baculoviruses carrying the 1-292, and cyclin D l, D2 or D3 cDNAs (lanes 6 -
8 ). 40 hour infected cells were metabolically labeled with L-[35S]methionine
and L-[35S]cysteine, and lysed with EBC containing 0.5% NP40 in the ratio
of 1.5xl06 cells/ml. 300 pi each of the lysates prepared from the cells
infected with the wild-type p53 (lane 1) and 1-292 (lane 4) viruses and from
the coinfected cells were combined with 0.2 pg of the antibody DO-1 and
precipitated with PAA (lanes 2-4 and 6 -8). 300 pi each of the lysates
prepared from the cells infected with the cyclin D l, D2, and D3 viruses were
mixed with 0.2 pg of the unrelated antibody DO-1 and PAA as negative
controls (lanes 9-11). The protein-antibody-PAA complexes were resolved on
10% SDS-PAGE. The calculated molecular weights of cyclins D l, D2, and
D3 are 33.67, 33.05, and 32.48 kD (Xiong et al., 1991 and 1992).
64
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wtp53 mP3C mP5C 1-366 1-351 1-342 66-393
D 2 - + - + - + . + . + . + - +
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fig. 2. Activating effect by deletion of either the C- or the N-terminus of
p53 on the p5 3-cyclin D interaction. How long of the minimal deletion from
the C-terminus of p53 will optimally activate p53-cyclin D interaction and
whether deletion of the N-terminus affects this interaction were tested by
coimmunoprecipitation. Sf9 cells were infected with individual recombinant
baculoviruses carrying the wild-type p53, mP3C, mP5C, 1-366, 1-351, 1-
342, or 66-393 cDNA (lanes 1, 3, 5, 7, 9,11, and 13), or coinfected with two
recombinant baculoviruses carrying the cyclin D2 and each of the viruses
above (lanes 2, 4, 6 , 8 , 10, 12, and 14). 40 hour infected cells were
metabolically labeled with L-[35S]methionine and L-[35S]cysteine, then
lysed with EBC containing 0.5% NP40 in the ratio of 1.5xl06 cells/ml. 300
pi of each lysate except the one containing 33-393 were combined with 0.2
pg of the antibody DO-1 and precipitated with PAA (lanes 1-12). 300 pi of
66-393 were combined with 0.2 pg of the antibody PAbl22 and precipitated
with PAA (lanes 13-14). The recovered proteins on PAA were resolved on
10% SDS-PAGE.
65
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enough to optimally enhance cyclin D2 binding (lane 10 versus 2, 4, 6 , and 8 ), and
deleting the N-terminal 65 residues also brought about a similar stimulating effect (lane
14 versus 2, 4, 6 , and 8 ).
To map the boundaries of the binding domain(s), more truncated p53 recombinant
baculoviruses were constructed and applied for testing. They included 1-211, 1-187, 1-
159, 1-99, 66-393, 100-393, 160-393, 237-393, and 293-393. The C- and N-terminal
boundaries for minimal binding activity were residue 159 (Fig. 3B, lane 6 ) and 237 (Fig.
3A, lane 6 ) respectively. 1-99 (Fig 3A, lane 4) with further deletion from 159 and 293-
393 (lane 8 ) from 237, lost the binding activity. The mapping result indicated two binding
domains existing. To determine the precise locations of the binding domains, three more
truncated p53 fragments coding for 100-225, 160-342, and 160-292, were constructed
into recombinant baculoviruses and applied for the further mapping experiment. All the
three mutants retained the cyclin D-binding activity (Fig. 3C, lane 1, 8 , 4, 9, 6 , and 10).
The final mapping result was presented in Fig. 4.: two binding domains lie within 1 G O -
159 and 237-292 and either one of them can interact with cyclin D independently.
Comparison of the Conformational Requirements for Sequence-Specific DNA
Binding and Cyclin D Binding by p53
Upon knowing that the two cyclin D-binding domains (100-159 and 237-292) lie within
the central core region where the DNA binding domain had been localized, we were
wondering if the p53/cyclin D interaction is also regulated similarly as the sequence-
specific DNA binding. The DNA- and cyclin D-binding profiles were compared. As
described in Chapter 2, either adding PAb421, mutation of P3 (Chapter 1, Fig. 7, lanes
10-12) or deletion of 27/more residues (Chapter 2, Fig. 5C, lanes) highly activated the
66
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-21
1 2 3 4 5 6 7 8 9 10 11
Fig. 3. Determination of the cyclin D-binding domains in p53 by coimmuno-
precipitation. Sf9 cells were infected with individual recombinant
baculoviruses or coinfected with combination of two viruses, which carry the
cyclin D2 or truncated p53 cDNA as indicated. 40 hour infected cells were
metabolically labeled with L-[35S]methionine and L-[3 5 S]cysteine, and then
lysed with EBC containing 0.5% NP40 in the ratio of 1.5xl06 cells/ml. 300 pi
of lysates were mixed with either anti-p53 antibody DO-1, PAb421, or rat anti-
cyclin D2 antibody Ab-1 as described below and precipitated with PAA. The
recovered proteins on PAA were resolved on 12% SDS-PAGE.
(A) Examination of the interactions between cyclin D2 and the truncated p53
mutants 1-99, 237-393, and 293-393. DO-1 was added in lanes 1-4, PAb421 in
lanes 5-8, and Ab-1 in lanes 9-11. (B) Examination of the interactions between
cyclin D2 and the truncated p53 mutants 1-211, 1-187, and 1-159. DO-1 was
added in lanes 1-6. (C) Examination of the interactions between cyclin D2 and
the truncated p53 mutants 100-225, 160-342, and 160-292. PAb240 was added
in lanesl-6 (epitope 212-217), and Ab-1 in lanes 7-10.
67
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Fig. 3 (continued)
-21
1 2 3 4 5 6
D2
D2
V
r t f ? r j C p ' '
+ - +
rfp 1 rjC p '
+ + + + +
-45
-31
-21
1 2 3 4 5 6 7 8 9 10
(B) Examination of the interactions between cyclin D2 and the truncated p53
mutants 1-211, 1-187, and 1-159. DO-1 was added in lanes 1-6. (C)
Examination of the interactions between cyclin D2 and the truncated p53
mutants 100-225, 160-342, and 160-292. PAb240 was added in lanes 1-6
(epitope 212-217), and Ab-1 in lanes 7-10.
68
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wt p53
CD(1-351)
CD(1-292)
CD(1-225)
CD(1-159)
CD(1-99)
100
J_ _ _ _
390
CND(100-225)
CND(160-292)
CND(160-236)
cyclin D2
binding
393aa
-/+
+
+
+
+
-
1 ND(66-393)
+
’ ND(100-393)
+
1 ND(160-393)
+
i ND(237-393)
+
1 ND(293-393)
+
+
?
^ The sequence-specific DNA binding domain
■■ The cyclin D binding domain
c=i The sequence that doesn't
contribute to the p53-cyclin D
interaction
Fig. 4. Summary of the mapping results for cyclin D binding
domains in p53 from Fig. 3(A), (B), (C), and unshown data
69
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latent p53 for binding to its responsive element. Deletion of the C-terminal 42/more
residues also enhanced binding of p53 to cyclin D2 (Fig. 2, lanes 10 and 12), whereas
mutation of P3 did not have obvious effect on this interaction (Fig. 2, lane 4). In addition,
the N-terminus of p53 also apparently participates in the regulation of these two events.
Deletion of the N-terminal 65 amino acid residues could enhance the cyclin D-binding
activity of p53 (Fig. 2, lane 14 versus lane 2); and modification of the N-terminus by
monoclonal antibody Pabl801 could activate the truncated p53, 1-289, for DNA binding,
which otherwise had lost this activity because of impaired integrity of the DNA binding
domain (Fig. 5B, lane 8 versus lane 6). However, unlike cyclin D binding, deletion of the
N-terminal residues alone (as shown in Fig. 5A) or binding by antibody Pabl801 (as
shown in Chapter 2, Fig. 5A and 5B) could not activate the latent p53.
The comparison of the two binding profiles has lead to a conclusion that different
conformation changes are required in order to expose or create the binding domains for
DNA and cyclin D.
Binding by Cyclin D Apparently Blocks the Truncated p53 from Forming a
Complex with DNA
Since the cyclin D-binding domains in p53 proved to be covered by the DNA-binding
domain, the question was asked: does binding p53 by cyclin D interfere with the p53-
DNA interaction? We performed EMSA with the p53 binding sequence BC and lysates
prepared from the truncated p53 and cyclin D2 recombinant baculoviruses coinfected
insect Sf9 cells (Fig. 6). No additional band from the nuclear extract containing both 1-
351 and cyclin D (lanes 7 and 8) was observed when compared with the shifting pattern
of that containing 1-351 only (lanes 5 and 6).
70
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PAb421 - +
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fig. 5. EMSA to examine the DNA binding activities of truncated p53
proteins. 5x109 of bacterial cells induced for expression of wild-type p53
and the truncated mutants were lysed in 200 pi of solution containing 50
mM HEPES, 10% sucrose solution and 0.5 mg/ml of lysozyme. 0.1 pg of
PAb421, DO-1, or equal volume of buffer was mixed with 1 pi of lysates
and incubated at 0°C for 30 min, then mixed with 0.1 ng of the 3 2 P-labeled
p53 binding sites containing DNA BC and continuously incubated for
another 30 min. The protein-DNA complexes were resolved on 4% PAGE.
(A) N-terminus truncated p53 proteins 39-393, 66-393, 100-393, 114-393,
and 160-393 were tested for DNA-binding capacities.
71
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Fig. 5 (continued)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
(B) C-terminus truncated p53 proteins 1-366, 1-292, 1-289, and 1-281
were tested for DNA-binding capacities
72
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DO-1 - + - + - + - + - + - +
D2 - - + + - - + + - -
wtp53_______ 1-292 1-351 BY
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 6. EMSA for testing the formation of the DNA-truncated p53-cyclin D
tripartite complex. Sf9 cells were infected with individual recombinant
baculoviruses or coinfected with two viruses as indicated. 48 hour infected
cells were lysed with EBC containing 0.5% NP40 in the ration of 4x107 cells
/ml. Antibody DO-1 was added to supershift the protein-DNA complexes.
The lysate prepared from the cells infected with wild-type baculovirus
provided the background of the binding reaction (lanes 11 and 12). 0.1 pg of
DO-1 or the equal volume of buffer was mixed with 1 pi of the lysates at 0°C
for 30 min. then mixed with 3 2 P-labeled BC for another 30 min. The protein-
DNA complexes were resolve on 4% PAGE. It was noticed that the wild-type
p53 proteins here were partially activated for DNA binding in the absence of
activating antibody PAb421 because of posttranlational modification in
eukaryptic cells.
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RB, Cyclin D3, and the Truncated p53 Barely Form a Tripartite Complex
The mapping data from the previous section showed that cyclin D physically
interacted with the partial of the central core region of p53, which includes domain II
(116-131), IV (234-258), and V (270-286) of the five highly conserved domains among
different species. This fact implies that the binding by cyclin D may be one of the
components of the p53 function. Furthermore, it was noticed in our experiments that the
anti-cyclin D3 antibody Ab-1 blocked the interaction between cyclin D3 and RB and
between cyclin D3 and truncated forms of p53 (data are not shown), we therefore
questioned whether RB and p53 compete with each other for the sam e binding domain or
two overlapping binding domains in cyclin D? Coimmunoprecipitation experiments were
conducted in which exclusion by each other between RB and p53 in forming a complex
with cyclin D was analyzed (Fig. 7). Lysates prepared from insect Sf9 cells infected with
a single, two, and three recombinant baculoviruses were used as th e sources of the tested
proteins. Fig. 7, lane 6 showed that Ab-1 precipitated cyclin D3 from the cell lysate
prepared from the cells coinfected with cyclin D3 and RB recombinant baculoviruses but
virtually none of the RB proteins was coprecipitated together. The complex containing
cyclin D3 and the truncated p53, 1-351, bound by antibody DO-1 did not bring down any
of the RB proteins (Fig. 7, lane 4), neither did the complex containing cyclin D3 and RB
bound by antiserum Ab2-3 bring down 1-351 obviously (lane 3).
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Ab 2-3 P16 2-3 DO-1 2-3 Ab-1
cycD3 - - + + + +
tsl-351 - - + + - -
RB + + + + + +
116-
97-
-RB
66-
45-
•1-351
m i: m * r n m + cycD3
31-
1 2 3 4 5 6
Fig. 7. Reciprocal exclusion between RB and the truncated p53 in the interaction
with cyclin D3. The competition between RB and p53 for binding cyclin D was
examined by coimmunoprecipitation. Sf9 cells were infected with single or two
recombinant baculoviruses , which carry the Rb, cyclin D3 or truncated temperature
sensitive p53 mutant tsl-351 cDNA as indicated. 40 hour infected cells were
metabolically labeled with L-[3 5 S]methionine and L-[3 5 S]cysteine, and lysed with
EBC containing 0.5% NP40 in the ratio of 1.5xl06 cells/ml. 300 pi of each lysate
were used for precipitation. As indicated, the RB proteins were precipitated with 2 pi
of the anti-RB serum Ab2-3 or 20 pi of the monoclonal antibody PI 6 (established by
graduate student Hong-Jun Zhang) containing hybidoma medium, the 1-351 proteins
with 0.2 pg of the antibody DO-1, and the cyclin D3 proteins with 0.2 pg of the rat
anti-cyclin D3 monoclonal antibody Ab-1. As it was known from previous
experiments that Ab-1 bound cyclin D3 fails to interact with both RB and p53, the
reaction in lane 6 was only designed to show the expression of this protein.The
antibody bound proteins were precipitated with protein G agarose (PGA) and
resolved on 10% SDS-PAGE.
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Discussion
The C-Terminus Truncated Forms of p53 Actively Interacting with Cyclin D Have
Similar Sizes to Those Natively Existing in Both Mouse and Human Tissues as
Translated Products of Alternatively Spliced mRNAs
Since the truncated form of p53 showed a much higher affinity for cyclin D than wild
type, we were wondering if this truncated form exist naturally in cells? Investigations
recently by several groups have showed the presence of alternative spliced form of p53 in
most mouse tissues (Han and Kulesz-Martin, 1992; Will et al., 1995) and all human
tissues (Flaman et al., 1996). The isoform of p53 mRNA from mouse cells encodes 1-364
amino acids from the normal spliced form (total 390 residues) plus 17 new amino acids
from intron 10; and the isoform from human cells encodes 331 amino acids from the
normal spliced form (total 393 residues) plus 10 new amino acids from intron 9. These
two isoforms of p53 are similar to the truncated mutants 1-366 and 1-341 used in our
experiments. Therefore, we reason that p53, in particular the alternative spliced form, can
interact with cyclin D in cells.
Differential Conformation Requirements by DNA and Cyclin D Bindings
Comparing the binding profiles of p53 to DNA and to cyclin D has revealed a
similarity: both the C- and the N-termini of p53 block its optimal interactions with DNA
and cyclin D. This implies existence of the mechanisms by which the interactions are
regulated. The comparison also suggests that the two regulatory mechanisms may be
different, all the truncated forms of p53 active for cyclin D binding are also active for
sequence specific DNA binding. However, there are mutations on the C-terminus which
dramatically increased the DNA-binding activity, but only barely enhanced the cyclin D
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binding activity. In addition, antibody Pab421 did not activate this interaction. This is
reminiscent of the role of p53 in in vitro replication assay with cell free extracts of
Xenopus eggs (Cox et al., 1995) in which Pab421 could not evoke the inhibitory activity
of p53 unless thirty amino acid residues were removed from the C-terminus. Our results
suggest that the conformations required for binding to DNA and to cyclin D may be
distinct from each other and indicate that the latter is more similar to the conformation
required for replication inhibitory function.
Functional Significance of p53-Cyc!in D Interaction: Being Regulated as a Target or
Regulating the Target?
p53-cyclin D complex apparently does not bind the p53 responsive elements. This
implies a regulatory function of D-type cyclins in p53-DNA interaction. Further
experiments involving competition with DNA to bind p53 with increasing amount of
cyclin D are needed. However, these experiments are difficult because p53 forms a stable
complex with cyclin D only when they are co-expressed in the same cell. The addition of
cyclin D to p53 in vitro does not lead to complex formation. Using the p53/cyclin D
complex purified with anti cyclin D antibody by affinity chromatography for this puipose
will be possible to overcome the difficulty. In this case, 100% of the purified truncated
p53 proteins will be in the complex with cyclin D, the effect of binding by cyclin D on
the p53-DNA interaction will be clearly observed.
The mutual exclusion between RB and p53 in binding to cyclin D binding leads to
another speculation that besides induction of transcription of p21, p53 might physically
block D-type cyclins from binding and phosphorylating RB whereby to more efficiently
stop the cell cycle in the G1 phase.
77
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However, our data are preliminary, therefore, the questions of which regulates which
and whether the two proteins regulate each other reciprocally can not be answered so far.
Further investigation is necessary in order to know the consequence of p53-Cyclin D
physical interaction.
The Interaction between p53 and D-Type Cyclins Has Been Proved by In Vivo
Experiment
Although the interaction between p53 and cyclin D was established inside of
eukaryotic cells, concentrations of the proteins with interest were definitely much higher
in our testing system than that under the physical conditions. Authenticity of the p53-
cyclin D interaction naturally in cells, therefore, needs to be proved. Very recently the
experiment conducted by Dr. Anne T ’ang shows that indeed endogenous p53 can be
coprecipitated with endogenous cyclin D from MCF7 human breast carcinoma cells
treated with UV light. This result has provided the in vivo evidence that p53 and cyclin D
physically interact with each other.
Conclusion
1. Cyclin D l, D2, and D3 interact with p53 and preferentially with the carboxyl- or
amino-terminus truncated forms. There are two cyclin D binding domains lie within 100-
160 and 237-292, overlapping the DNA-binding domain of p53.
2. The conformation requirement for p53 to interact with cyclin D is more similar to
that required for the inhibition of DNA replication than to that required for DNA binding.
3. Binding by cyclin D apparently blocks p53 from binding its responsive element.
78
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4. The cyclin D protein complexed with p53 is lack of the ability to interact with RB,
and vice versa.
Materials and Methods
Plasmids and Baculoviruses
pETwtp53 and the mutant plasmids used for mapping the sequence-specific DNA
binding domain in EMSA were as same as those described in Materials and Methods of
Chapter 2. Plasmids pACYMlp53, its mutants and pACYMlRb were used as cDNA
providers and Bsu36 I digested wild-type baculovirus Bakpack 6 genomic DNA as a
recipient for construction of the corresponding recombinant baculoviruses by transfection
and in vivo recombination. Preparation of the viral genomic DNA followed the protocol
in A Manual of Methods for Baculovirus Vectors and Insect Ceil Culture Procedure
by Max D. Summers and Gale E. Smith. Cotransfection of the plasmid and viral DNA
was performed by the method of lipofection. Host cell strain for receiving DNAs was Sf9.
5x 105 Sf9 cells from a rolling culture in TNM-FH medium containing FBS and
antibiotics were plated in each 35 mm cell culture dish and grown overnight a t 26°C. 2 pg
of the plasmid and 1 pg of the viral DNA were mixed and diluted to 100 pi with ddH20;
15 pi of lipofectin (GIBCO BRL) were diluted to 100 pi with ddH20. The two solutions
were mixed well and placed at room temperature for 20-30 min for DNA-lipofectin
combination. At the same time, the cells in the dishes for transfection were rinsed for two
times with FBS free TNM-FH medium. The cells were incubated in the sam e medium at
room temperature until DNA-lipofectin combination was over. 800 pi of the same
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medium was added to each DNA-lipofectin solution and gently mixed with a pipet. The
medium-DNA-lipofectin solutions were overlaid on the cells after removing the medium
from the dishes. Lipofection lasted overnight (about 12 hrs) at 26°C and the medium was
replaced the next morning with a regular TNM-FH medium containing FBS and
antibiotics. The transfected cultures were incubated at 26°C until an obvious infectious
phenomenon occurred (about 6-7 days posttransfection). Viruses released into medium
were collected and stocked in the medium at 4°C in darkness. Viruses were ready for
experiment after amplification for one time by infection of new Sf9 cells with primary
virus stocks.
The cyclin D l, D2 and D3 recombinant baculoviruses were kindly provided by Dr.
Charles Sherr.
Immunoprecipitation
4xl06 Sf9 cells from a rolling culture in TNM-FH medium containing FBS and
antibiotics were plated into a T25 flask. The medium was replaced after 1 hr attachment
at 26°C and the cells were grown in the flask overnight at the same temperature. For
testing p53-cyclin D interaction, the overnight growing cells were infected with one or
two recombinant baculoviruses, i.e. cyclin D, p53, and the mutant virus only, and cyclin
D virus combined with p53 or the mutant virus at 26°C for 1-2 hrs. The medium was
replaced after infection and the culture was continuously incubated for 40 hrs. For testing
p53-cyclin D-RB interaction, cells were coinfected with three baculoviruses otherwise the
operations were as same as above.
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Infected cells were knocked off from surface of the flasks, pelleted by centrifugation
in an DEC clinical centrifuge at speed grade 3 for 2 min at room temperature, rinsed with
L-methionine free RPMI 1640 medium (GIBCO BRL), and resuspended in the same
medium containing 5% dialyzed FBS in the ratio of 1 x 106 cells/0.5 ml. Expre3 5 S3 5 S was
added to the cells in the ratio of 50 pCi/5 x 105 cells. Labeling was conducted at 26°C for
3-4 hrs.
Labeled cells were transferred into eppendorf tubes and pelleted by centrifugation at
6,000 rpm for 3 min at room temperature. The pellets were rinsed with PBS, pH 7.4,
briefly. The cells were suspended in EBC lysis buffer (20 mM Tris-HCl, pH 8.0, 125 mM
NaCl, 0.5% NP40, 2 mM EDTANa2, phosphatase inhibitors: 20 mM NaF and 0.1 mM
Na3VC>4, and proteinase inhibitors: 1 pg/ml aprotinin, 1 pg/ml leupeptins, 1 pg/ml
pepstatin A, 0.1 mM PMSF) in the ratio of 5 x 105 cells/300 pi and kept on ice for 1 hr.
The lysates were centrifuged at 14k rpm for 10 min at 4°C. 300 pi of each supernatant
was mixed with antibody against one of the tested proteins and rocked at 4°C for 1 hr. 15
pi of PAA or PGA (protein G agarose) larry saturated with BSA was added to the mixture
and continuously rocked for 1 more hr. The protein-protein-antibody complexes bound to
PAA or PGA were rinsed with 1 ml of cold EBC (20 mM Tris-HCl, pH 8.0, 125 mM
NaCl, 0.5% NP40, 2 mM EDTANa2, and phosphatase inhibitors: 20 mM NaF and 0.1
mM Na3V 0 4 ) and pelleted in a microfuge at 12k rpm briefly, EBC was removed by
vacuum. Rinse-pellet-vacuum was repeated for 2 more times. The pellets were suspended
in 15-20 pi of 2x loading buffer and heated at 86°C for 10 min. The rest steps including
81
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SDS-PAGE electrophoresis, gel treatment and visualization of result were as same as
described in the section of Immunoprecipitation in Materials and Methods of Chapter 2.
The Gel Electrophoretic Mobility Shift Assay (EMSA)
1. Determination of the sequence-specific DNA binding domain of p53. p53 and its
truncated mutants proteins were expressed in E. coli strain BL21. The procedures for
lysate preparation, probe labeling, p53-DNA binding reaction, and electrophoresis were
as same as described in the section of The Gel Electrophoretic Mobility Shift Assay in
Materials and Methods of Chapter 2.
2. Examination of cyclin D-truncated p53 complex for its DNA binding activity. The
sources of p53 and cyclin D proteins were from whole cell lysate of recombinant
baculoviruses infected Sf9 cells. Cells were infected with single recombinant baculovirus
or combination of recombinant baculoviruses carrying the p53, its mutant and cyclin D
cDNAs for 1 hr at 26°C. The medium was changed after infection. The cells were
incubated at the same temperature for 48 hrs, then collected, rinsed with PBS, pH7.4, and
lysed with EBC lysis buffer. The procedures for probe labeling, p53-DNA binding
reaction, and electrophoresis were as same as described in the section of The Gel
Electrophoretic Mobility Shift Assay in Materials and Methods of Chapter 2.
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CHAPTER 4: INVESTIGATION OF FUNCTIONAL SIGNIFICANCE
OF p53-RBP2 INTERACTION AND EXPLORATION OF RBP2
FUNCTION
Summary
The study described in Chapter 2 provided the evidence that P3 (372-380) is part of
the motif controlling the p53-DNA interaction. We were wondering then whether any
other cellular proteins, besides TBP, interact with p53 and regulate its DNA-binding or
other functions through targeting P3. At the same time, we also started searching for
cellular proteins that are physically associated with p53 by coimmunoprecipitation.
Preliminary data revealed an interaction of RB-binding protein-2 (RBP2) with p53 in
vitro. To confirm this interaction and understand its functional significance, we have
localized the binding domains in both p53 and RBP2, tested the requirement of P3 for
p53-RBP2 interaction, examined the activity change of p53 in both DNA binding and
transcriptional activation in the presence of RBP2, and explored the function of RBP2.
Three RBP2-binding domains in p53 were determined. The C-terminal binding
domain lies within the protein sequence 325-380 and it controls the other two upstream
binding domains, 66-225 and 237-342, in the p53-RBP2 interaction. Two p53-binding
domains in RBP2 were also determined. The downstream domain (1458-1599) overlaps
with one (1457-1558) of the two RB-binding domains and the TBP-binding domain
(1457-1558) (Kim et al., 1994).
83
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Similar to the case of p53-TBP binding experiment described in Chapter 2, among all
the missense mutations tested was the P3 mutation that most completely eliminated the
binding activity of p53 to RBP2/1200-1722.
Since RBP2 directly interacts with the p53 C-terminus (325-380) and P3 (372-380) is
absolutely required for the interaction, we tested whether binding by RBP2 had any effect
on the sequence-specific DNA binding capacity of p53 by EMSA. However, we found
that RBP2 itself had a high affinity for double-stranded DNA. The DNA-binding domain
of RBP2 was mapped to a region that overlaps with the p53-, RB-, p i30, and TBP-
binding domains. Having defined the DNA binding domain (1458-1599), which overlaps
with but longer than the minimal p53-binding domain 1497-1599, we re-examined the
effect of binding by RBP2 on the DNA-binding capacity of p53 with a truncated RBP2
(1497-1722) which has lost the ability to bind DNA but still retains the ability to bind
p53. The result showed that this mutant could not activate the DNA binding by p53 in
vitro. To find out if RBP2 can enhance p53 transcriptional transactivity in cells, a
transient transfection-transcription assay was performed. Consistent with the lack of
effect on the DNA binding capacity of p53, cotransfection with the full length of RBP2
cDNA did not incur an obvious change in p53 transcriptional transactivity when
compared with transfection of the p53 cDNA alone under the conditions when the cells
were actively growing. Neither did cotransfection of RBP2 affect the transcriptional
repression function of p53.
Thus, based on the data obtained, RBP2 apparently doesn’t affect the p53
transcriptional regulation function. To address the second question of whether RBP2-p53
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interaction affects RBP2 function, we turned to exploring the function of RBP2. Since
had demonstrated RBP2 to be a double-stranded DNA binding protein, and the binding
domain overlaps with its p53-, RB- and TBP-binding domains, we asked whether RBP2
has a function in transcription or growth regulation. We tested transactivity of full length
RBP2 and its truncated forms as GAL4 chimeric proteins to the GAL4 binding sites-
containing promoter in mammalian cells. No transcriptional transactivity was detected by
this experiment. To evaluate the contribution of RBP2 to growth regulation, we compared
the effects on cell growth of overexpression of wild-type RBP2 and a RBP2 mutant with
deletion of the overlapping region among several domains interacting with RB family
members, p53, and DNA. Location of the expressed mutant RBP2 was detected in the
cytoplasm of transfected cells in contrast to the nuclear location of the wild-type protein.
A much higher percentage of 2-day posttransfected cells, expressing the mutant was
observed than that expressing the wild-type RBP2. H358 tumor cells transfected with
wild-type RBP2 formed less and smaller colonies than with the mutant when examined
30 days posttransfection. We therefore speculate that the cellular function of RBP2 may
relate with growth arrest or cell death.
Introduction
Genetic Information and Structure of RBP2
RBP2 is a phosphonuclear protein with 1722 amino acids (Fattaey et al., 1993). It was
first isolated from a lambda gtl 1 human cDNA expression library as a RB pocket domain
associated peptide (Defeo-Jones et al., 1991). The gene is expressed ubiquitously in
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different tissues and is conserved among different species including monkey, mouse, cow,
chicken and rabbit (Defeo-Jones et al., 1991). Computer search (Fattaey et al., 1993)
showed that RBP2 has a putative zinc finger structure sharing homology with the general
transcription factor TFHE, and that two regions of RBP2 also have sequence similarity to
the homeodomain of the engrailed family of homeotic genes. So far no protein derived
from cDNA in GenBank has shown extensive similarity to RBP2.
RBP2: a Function-Unknown Protein
Comparison of the sequences between RBP2 and DNA tumor viral oncoproteins
uncovered that one of the two Rb-binding domains on RBP2 shares a homologous
sequence including LXCXE with El A, simian virus 40 large T antigen (large T) and
human papillomavirus 16 E7 (Defeo-Jones et al., 1991; Dyson et al., 1989). The
homologous sequence is necessary and sufficient for the interaction of large T with RB
and its transforming potential (DeCaprio et al., 1988 and 1989; Ludlow et al., 1990); The
collinear sequences in E l A and E7 are also necessary to their RB-binding and
transforming activities (Dyson et al., 1989; Green 1989; Whyte et al., 1989).
E2F, a family of transcription factors, was first described as a cellular activity
responsible for ElA-dependent activation of adenovirus E2 promoter (Kovesdi et al.,
1986), and E2F1 was first cloned as a RB-associated protein (Helin et al., 1992; Kaelin et
al., 1992; Shan et al., 1992). A substantial group of the genes required for DNA
replication and cell cycle progression were identified to possess the E2F binding sites and
many of them have been demonstrated to be up-regulated by E2F such as c-myc (Oswald
et al., 1994), dihydrofolate reductase (DHFR) (Slansky et al., 1993), B-myb (Lam and
86
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Watson, 1993), DNA polymerase-alpha (Sala et al., 1994), cyclin D1 (Sala et al., 1994),
cdc2 (Degregori et al., 1995), cyclin A (Schultze et al., 1995), cyclin E (Ohtani et al.,
1995; Botz et al., 1996), PCNA (Yamaguchi et al., 1995), H2A (Oswald et al., 1996), and
E2F1 itself (Neuman et al., 1994). When over-expressed, E2F can induce quiescent cells
to enter S phase (Johnson et al., 1993; Qin et al., 1994; Schwarz et al., 1995). The
underphosphorylated form of RB binds E2F (Chellappan et al., 1991; Kaelin et al., 1992)
and represses its transcriptionally activating the target genes (Hiebert et al., 1992;
Hiebert, 1993; Flemington et al., 1993; Cress et al., 1993; Helin et al., 1993). E1A, Large
T, and E7 can release E2F from the repression by dissociating the RB-E2F complex
(Chellappan et al., 1991; Bandara and La Thangue, 1991; Chellappan et al., 1992;
Zamanian and La Thangue, 1992). Studies have shown that like large T (Ludlow et al.,
1989), E7 (Imai et al., 1991) and E2F (Chellappan et al., 1991), RBP2 preferentially
interacts with the underphosphorylated RB protein, and when cotransfected with the Rb
cDNA, releases E2F-dependent transcription repressed by RB (Kim et al., 1994). RBP2
also interacts directly with TBP (Kim et al., 1994). Existence of the LXCXE containing
homologous motif, its interaction with the underphosphorylated RB protein, and apparent
antagonization with the RB protein in transcription assay have lead to the speculation that
RBP2 might be a cellular counterpart of the DNA tumor viral oncoproteins.
Very recently human SWI2/HFN2 homologues, hBRGl and hBrm, were found also to
be LXCXE carrying proteins (Dunaief et al., 1994; Singh et al., 1995). Yeast
Saccharomyces cerevisiae SWI2/SNF2 is a protein essential for regulating the expression
of a variety of genes (Peterson and Herskowitz, 1992; Yoshinaga et al., 1992). Members
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of SWI2/HFN2 family do not bind DNA directly. Some of them have a proline-glutamine
rich activation domain. Enhancement of transcription by these proteins is achieved by
their physical interaction with other DNA binding transcription factors. hBrm is such a
transcription coactivator which binds the glucocorticoid receptor (GR) and potentiates its
transcriptional activation (Muchardt and Yaniv, 1993; Singh et al., 1995). Like DNA
tumor viral oncoproteins E1A, Large T, and E7 and cellular protein RBP2, hBRGl and
hBrm interact preferentially through their LXCXE motif-containing region with
hypophosphorylated RB. Studies showed that by binding to RB, hBRGl could induce
growth arrest of hBRGl-negative adrenal carcinoma SW13 cells (Dunaief et al., 1994),
and that cotransfection of hBrm with Rb cDNA further potentiated GR-dependent
transcription (Singh et al., 1995). Since the Rb gene has been proved to function in tumor
suppression (Huang et al., 1988; reviewed by Riley et al., 1994), embryo development
(Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992; Riley et al., 1994), and cell
differentiation (reviewed by Riley et al., 1994, and by Chen et al., 1995), hBrm is
possibly a factor involved in any of these RB-dependent biological events. These results,
together with the fact that RBP2 possesses homeodomain homologous sequences (Fattaey
et al., 1993), lead to the speculations that RBP2 might act in cooperation with RB like
hBRGl and hBrm, and that the viral oncoproteins might promote growth and transform
cells partially by competing to bind hypophosphorylated RB and whereby abrogating its
interactions with the cellular LXCXE containing proteins.
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Discovery of an Interaction between RBP2 and p53
As described in Chapter 1, several kinases including CK-II, PKC, Cdk2, and Cdc2
phosphorylate p53 and activate its DNA binding in vitro (Hupp et al., 1992; Wang and
Prives, 1995). However, no one had reported any cellular protein that can activate p53 by
targeting its carboxyl-terminus regulatory domain. We were thus greatly interested in
looking for any such candidate(s). At the same time, we noticed that both RB and p53 are
growth suppressors, and that accumulated evidence indicated p53, like RB, also being
involved in cell differentiation (Shaulsky et al., 1991; Feinstein et al., 1992). We were
wondering whether RB and p53 interact with the same cellular proteins and, through
them, cooperate with each other. We were inspired by this consideration to choose
cellular proteins which associate with RB as a pool of inspected proteins when we started
searching the candidates for the regulators of DNA binding activity of p53. Three out of
the tested proteins, RBP2 (by Ling Li), PU.l (by Dr. Chen Li) and MyoD (by Dr. Chen
Li) were found to bind p53 in the coprecipitation experiment.
Identification of RBP2, PU.l, and MyoD as p53-binding proteins raised questions: do
these proteins mediate p53 sequence-specific DNA binding and further transcriptional
regulation, or does p53 regulate these proteins? As RBP2 was the first to be identified as
a p53-binding protein among the three, we started with investigating the relation between
RBP2-p53 interaction and p53 function. And as little was known about the RBP2
function, in order to investigate the effect elicited by p53 on the function of RBP2 in
future, we explored the function of RBP2. This chapter described the findings and results
from our research: p53 interacts with RBP2 in vitro through three binding domains, RBP2
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is a double-stranded DNA-binding protein, nuclear localization signal resides within the
region 1497-1599, and overexpression of wild-type RJBP2 suppresses colony formation of
H358 tumor cells. The significance of p53-RBP2 interaction is being investigated.
Results
Localization of the RBP2-Binding Domains in p53 and the p53-Binding Domains in
RBP2
To find out whether RBP2 directly interact with the C-terminus of p53, we mapped
the RBP2-binding domains in p53 by coprecipitation. The p53 and mutant proteins
expressed in either bacteria or by baculoviruses, were coprecipitated with immobilized
GST-RBP2/1200-1772 on GS-4B. The binding domains were localized within 66-225,
237-343 and 325-380 (Fig. 1).
The following RBP2 binding profiles were noticed in the experiment: deformation of
the tetrameric structure of p53 by mutation of PI (343-351) doesn’t affect the interaction
between p53 and RBP2/1200-1722 (Fig. 2, upper panel, lane 3); three mutants mP4 (the
DNA binding domain mutated), mP5C (conserved residues within 381-393 mutated), and
1-380 (P5 deleted), which failed to bind DNA, have a very similar affinity to the wild
type for RBP2/1200-1722 (lanes 2, 7, and 9 versus lane 1); mP5U carrying mutation of
the unconserved amino acid residues within P5 (381-393) is more active for RBP2
binding compared with wild-type (lane 8 versus lane 1); and the DNA binding active
mutants including mP2 (residues 357-370 selectively mutated) (lane 4), mP3C (conserved
residues within 372-380 mutated) (lane 5), and the C-terminus truncated forms, 1-366 and
1-342 (Fig. 1), are defective in binding RBP2 despite the other two upstream RBP2-
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wtp53
100 DNA binding 300
RBP2 /
1200-1722
binding
393
mP5 (381-393)
mP3(372-380) 1...... — ■
mP2(357-370)
l ' T l
mPl (343-351)
CD(1-3 80)
CD(1-3 66)
CD(1-342)
CD(1-292)
CD(1-187)
CD(1- 99)
ND( 66-393)
ND(100-393)
ND(160-393)
ND(237-393)
ND(293-393)
ND(325-393)
NCD(293-383)
NCD(293-368)
NCD( 66-225)
NCD(160-342)
RBP2-binding
domains:
K \\\v\\vv\\^\\v^\\vvv\\\vvv\V N .\\v\\\\\vvvv\\vv\\v\vvvV vvvvvvv\yvvj
66-225 237-342
+ ++
+ ++
+ / -
+ / -
+++ +
+++
+ +
++
325-380
Fig. 1. Localization of RBP2-binding domains in p53. The filled and
hatched bars above represent mutants with different affinities for
truncated RBP2, and the blanked bars mutants defective for
binding. Names for the mutants are on the left side of bars:
mP=missense mutant, CD=C-terminus deletion mutant, N D = N -t
erminus deleion mutant, and N C D =N - and C-term ini deletion
mutant. Numbers in the brackets behind the names indicate the
locations of mutated peptides in p53 or the sizes of deletion
mutants. The black bars represent three defined RBP2-binding
domains and numbers beneath their locations in p53.
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O ID O D O
1 2 3 4 5 6 7 8 9
Fig. 2. Mutation at P3 (372-380) or P2 (357-370) abrogates the interaction
between p53 and the C-terminus of RBP2. The interactions of the C-terminus of
RBP2 with p53 and the mutants were examined by coimmunoprecipitation. p53
protein and its mutants were expressed in bacteria and metabolically labeled with
L-[3 5 S]methionine and L-[3 5 S]cysteine. GST-RBP2/1200-1722 chimeric protein
was expressed in bacteria without labeling. The bacteria were aliquoted, pelleted,
and frozen at -80°C. The protein expression was proved and the protein quantities
were examined in lower panel before the experiment in upper panel was
conducted.
Upper panel, copricipitation of wild-typw p53 and the mutants with 1200-1722 of
RBP2. 5x108 of the frozen cells were lysed in 100 pi of PBS containing 0.25
mg/ml lysozyme. The GS-4B bound 1200-1722 chimeric proteins prepared from
25 pi of lysate were mixed with 25 pi of lysates containing the wild-type or
mutant p53 proteins as indicated. The unbound proteins and impurities were
removed by rinsing the GS-4B beads with EBC. The proteins precipitated were
resolved on 8% SDS-PAGE. Lower panel, Equalization of the p53 and mutant
proteins by immunoprecipitation. 5xl08 of the frozen cells were lysed in 100 pi
of PBS containing 0.25 mg/ml lysozyme. 25 pi of the lysates containing the
proteins to be tested were combined with 0.3 pg of the antibody DO-1 and
precipitated with PAA. Thus the same number of cells was used in each
precipitation in the upper panel.
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binding domains are intact (lanes 4 and 5). Coprecipitation experiments using GST fusion
proteins containing the C-terminal aa 293-393 and other truncated forms 293-383, 293-
368, and 325-393, fused to GST provided further evidence that the deletion of 357-380
was enough to abrogate the RBP2 binding activity of the C-terminal domain (Fig. 1).
Effect of binding by two anti-P3 antibodies PAb421 and PAbl22 on the p53-
RBP2/1200-1722 interaction was also tested by coprecipitation (Fig. 3). Compared with
antibody DO-1 (Fig. 3, lane 4) directed to the N-terminal residues 21-25, the binding
capacities of both PAb421 and PAbl22 bound p53 to RBP2/1200-1722 were greatly
decreased (lanes 2 and 3).
Two p53-binding domains in RBP2 were also determined by coprecipitation.
Different cDNA fragments created by PCR were fused to GST to construct a ray of
chimeric deletion mutants encoding the truncated RBP2 1200-1722, 1200-1599, 1200-
1496, 1200-1457, 1458-1722, 1497-1722 and 1593-1722. Two p53 proteins, the wild type
and the truncated mutant 66-225, were used for this purpose. Two p53-binding domains
were defined, using these truncated proteins, within 1200-1496 and 1497-1599 of RBP2
as shown in Fig. 4.
RBP2 Is a Non-Specific Double-Stranded DNA-Binding Protein
Since RBP2 was primarily demonstrated to bind p53 and one of the three RBP2-
binding domains detected within 325-380 overlaps with the negative regulatory domain
for sequence-specific DNA binding activity of p53, a question was raised: Does binding
by RBP2 stimulate this activity? EMSA was performed to answer the question.
Unexpectedly, RBP2/1200-1722 itself could bind to the p53 binding consensus sequence
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Ab 421 122 DO-1 421 122 DO-1
1200-1722 + + + + - - -
p53 - + + + + + +
116-
1 2 3 4 5 6 7
Fig. 3. Binding the RBP2 C-terminus 1200-1722 by p53 is blocked by anti-p53
P3 antibodies PAb421 and PAbl22. Competition for binding P3 between
PAb421/PAbl22 and the C-terminus of RBP2 was demonstrated by
coimmunoprecipitation. GST-RBP2/1200-1722 and p53 were expressed in
bacteria and metabolically labeled with L-[3 ;,S]methionine and L-[3 :iS]cysteine.
5x108 of both bacteria were lysed with 100 pi of PBS containing 0.25 mg/ml
of lysozyme. 0.2 pg of the antibody DO-1, PAb421 and PAbl22 was mixed
with 25 pi of the lysate containing the p53 proteins and precipitated with PAA
as indicated. Two repeats of each sample above were prepared. After rinsing
the protein-antibody-PAA complexes with EBC, one of the repeats was ready
for electrophoresis (lanes5-7), the other was mixed with 25 pi of the lysate
containing the GST-RBP2/1200-1722 proteins (lanes 2-4). The unbound
proteins and impurities were removed by rinsing the complexes with EBC.
The GST-RBP2/1200-1722 proteins in lane 1 were precipiated with GS-4B
from 100% input of the lysate. The precipitated proteins were resolved on 8%
SDS-PAGE. DO-1 is directed to residues 21-25, and PAb421 and PAbl22 to
residues 370-380 of p53.
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truncated RBP2
fused to GST
1200-17 22
1200 -1 5 9 9
1200 -1 4 9 6
1 200-14 57
1 458-17 22
1 497-17 22
1593 -1 7 2 2
L X C X E
p53
full length
++
+ +
++
++
p53
66-225
?
+
p53-binding
domains
1200 -1 4 9 6 1497-1599
F ig . 4. Lo calization o f p53-binding domains in R B P 2. Seven
o f eight bars from the top represent d iffe re n t truncated
R B P 2 peptides, and numbers on th e ir le ft side pepside
sizes. The bar at the bottom is the summary fo r m apping
resu lt. The black c o lo r-fille d bigger areas represent the
dom ain carryin g the binding a c tiv ity fo r the fu ll-le n g th
p 5 3 , and the hatched areas the dom ain c a rry in g the
b inding a c tiv ity fo r the truncated p53, 6 6 -2 2 5 . Q uestion
m arks represent the in teractions w hich have not been
e x a m in e d .
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BC used in the assay. To determine if the binding was sequence specific or non-specific,
another double-stranded DNA containing 2 repeats of E2F-binding site was replaced for
BC (compare Fig. 5, lanes 6-10, with lanes 1-5). Identity of the binding protein was
proved by anti-GST monoclonal antibody (lanes 4 and 9). An unrelated anti-p53 antibody
DO-1 was arranged as a negative control (lanes 5 and 10). The result shows that 1200-
1722 binds both DNAs. The binding intensity is protein dose dependent (Fig. 5, lanes 1-3
and 6-8). In addition, a competition experiment was performed to further prove the non
specificity of the protein-DNA interaction by EMSA (Fig. 6). The binding to the 3 2 P-
labeled BC and E2F DNAs by RBP2/1200-1722 could be competed away by increasing
amount of the same or different cold DNA.
The DNA-binding domain on RBP2 was localized (in cooperation with Dr. Chen Li)
by EMSA with the same range of the GST-truncated RBP2 fusion proteins used in the
experiment for determining the p53-binding domains. The binding domain was defined
within 1458-1599 a s shown in Fig. 7.
RBP2/1497-1599 Does Not Stimulate Sequence-Specific DNA Binding of p53 In
Vitro
Since RBP2/1200-1722 was bound to both p53 and DNA, it was impossible to use
this truncated RBP2 for testing its effect on the DNA-binding capacity of p53 because of
DNA binding competition between the two proteins. Localization of the DNA-binding
domain on RBP2 within residues 1458-1599 allowed us to use the domain 1497-1599,
which was able to bind p53 but not DNA for EMSA. The result showed that binding by
this truncated protein did not activate p53 for sequence-specific DNA binding (Fig. 8).
Other than p53 molecules inadvertently activated during the protein purification
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RBP2
Fig. 5. The C-terminus of RBP2 binds double-stranded DNA in a sequence-
non-specific manner. The DNA binding property of the RBP2 C-terminus
was demonstrated by EMSA. Bacteria expressed GST-RBP2/1200-1722
fusion proteins were purified by affinity chromatography with GS-4B. 150
ng of the mouse anti-GST monoclonal antibody A bl2 were added to and
incubated with 240 ng of the GST-RBP2 protein in lanes 4 and 9 or 240 ng
of the GST protein in lanes 12 and 14 at °C for 30 min before addition of
DNA. 150 ng o f the unrelated antibody DO-1 were operated as same as
A bl2 in lanes 5, 10, and 13. 0.1 ng of the 3 2 P-labeled p53 binding sites
containing D N A BC or E2F binding sites containing D N A E2F as indicated
was mixed with increased amount o f the GST-RBP2/1200-1722 protein, 60
ng (lanes land 6), 120 ng (lanes 2-7), and 240 ng (lanes 3-5, and 8-10) and
continuously incubated for another 30 min. The protein-DNA complexes
were resolved on 4% PAGE.
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Cold probe
3 2 P-probe BC
E2F BC
E2F
*
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 6. Competition for binding the C-terminus of RBP2 between 3 2 P-labeled
and cold DNA fragments with same or different sequences. EMSA to further
prove that the DNA binding by the C-terminus of RBP2 is sequence non
specific was performed. 250 ng of the affinity chromatography purified GST-
RBP2/1200-1722 protein waas mixed and incubated with increased amount
of the cold p53 binding sites containing DNA BC or the E2F binding sites
containing D NA E2F, 0 ng (lanes 1 and 8), 2.5 ng (lanes 2 and 5), 12.5 ng
(lanes 3, 6, 9, and 11), and 37.5 ng (lanes 4, 7, 10, and 12) at 0°C for 30 min,
then mixed with 0.1 ng of the 3 2 P-labeled BC (lanes 1-7) or E2F DNA (lanes
8-12) and continuously incubated for another 30 min. The protein-DNA
complexes were resolved on 4% PAGE.
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truncated RBP2
fused to GST
DNA binding
L X C X E
1200-1722 |... ............... 1-------
1 20 0 -1 5 9 9 , --------------
1 20 0 -1 4 9 6 i =
1 200-14 57 i -------
1458-17 22
1497-1722
1593-17 22
DNA-binding
domain: 1458-15 99
F ig . 7. Schem atic presentation o f R BP2 deletio n m utants
and the corresponding D N A -b in d in g a c tiv itie s . Seven bars
fro m the top represent tru n cated R B P 2 -G S T fu s io n
peptides. Num bers on the le ft side o f the bars sizes o f
truncated R B P2. B lack c o lo r-fille d bigger areas in the bars
represent the dom ain w hich in teracts w ith D N A . The
black bar at the bottom is the sum m ary o f the m apping
result o f D N A binding activity.
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procedure which formed a complex with DNA and caused a shift, there is neither increase
in the binding nor is there supershift in the presence of both p53 and GST-RBP2/1497-
1599 (lane 3) compared with in the presence of p53 only (lane 1).
No Effect of Coexpression of RBP2 Detected on the Transcriptional Activation and
Repression Functions of p53
The question of whether RBP2 stimulates transactivity of p53 was addressed by a
transient transfection-transcription assay. The reporter POSTCAT2 (described in Chapter
2) and expression plasmids carrying the p53 and RBP2 cDNAs were introduce into H358
cells (p53-/-) by electrooporation. RBP2/1-1201, a mutant with deletion of RB-, p53- and
DNA-binding domains was applied a s a negative control to wild-type RBP2. The effector
and control cDNAs are transcriptionally controlled by the cytomegalovirus immediate
early promoter/enhancer. Consistent with the EMSA data, expression of RBP2 does not
affect transcriptional transactivity of p53 when cotransfected with the p53 cDNA (Fig. 9,
lanes 3-5 versus 2).
Effect of coexpression of RBP2 on transcriptional repression function of p53 was also
tested by the same assay. pSV2CAT, provided by Dr. Bruce Howard (Gorman et al.,
1982), was used as a reporter which comprises a structure of the SV40 early gene
promoter driving CAT gene. p53 was reported to repress transcription from this promoter
(Jackson and Braithwaite, 1993). The same plasmids carrying the p53 and RBP2 and
RBP2 mutant cDNAs as described in the transcriptional activation assay above were
used. DNAs were introduced into Hela cells by electroporation. The transcriptional
repression function of p53 was not affected by coexpression of RBP2 (Fig. 10, lanes 5
and 6 versus lane 1).
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GST-RBP2/
PAb421 1497-1599
GST-p53 + + +
12 3 4
Fig. 8. The truncated RBP2 1497-1722 does not stimulate the sequence-
specific DNA binding activity of p53. The effect of adding the truncated RBP2
1497-1722 on the sequence-specific DNA binding capacity of p53 was
investigated by EMSA. 0.1 pg of the affinity chromatography purified GST-
p53 chimeric protein was mixed and incubated with buffer only (lane 1), 0.1
pg of the antibody PAb421 (lane 2), or 0.5 pg of the same method purified
GST-RBP2/1497-1722 chimeric protein (lane 3) at 0°C for 30 min, then mixed
with 0.1 ng o f the 3 2 P-labeled p53 binding sites containing DNA BC and
continuously incubated for another 30 min. The protein-DNA complexes were
resolved on 4% PAGE.
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Fig. 9. Coexpression of RBP2 doesn’t change the transcriptional transactivity
of p53. The effect of coexpression of RBP2 on the transactivity of p53 was
examined by the transient transfection assay. H358 cells (p53-/-) were
cotransfected with 2.5 pg of the POSTCAT2 reporter, 2.5 pg of the p53
expression plasmid and increased amount of the wild-type RBP2 expression
plasmid, 0 pg (lane 2), 12.5 pg (lane 3), 25 pg (lane 4) and 50 pg (lane 5).
Trnasfection was also performed b y replacing the deletion mutant 1-1201 for
the wild-type RBP2 plasmid (lanes 6-8). 1-1201 is a mutant with deletion of
the RB, p53, and DNA-binding domains. The background was provided in
lane 1 by transfering cells with 2.5 pg of the reporter only. The transfected
cells were harvested 60 hour posttransfection and lysed by freeze-thaw cycles.
Total protein concentrations of lysates were measured with Bio-Rad protein
assay dye reagent. The endogenous acetyl transferase activity was heat-
inactivated at 65°C for 10 min. Enzyme reactions were conducted with the
same amount of total proteins from the lysates, in the presence of 0.125 pCi of
D-threo-[dichloroacetyl-l-1 4 C]chloramphenicol and 1 pi of 40 mM acetyl
coenzyme A. About one third of each lysate was used for the assay. The
products and unreacted substrates were extracted from the reaction mixtures
with acetyl acetate, dried, resuspended in a small volume of acetyl acetate, and
resolved on a silica gel coated plate by thin layer chromatography.
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P53 - - - + + + + +
$ 4 . ^
1 2 3 4 5 6 7 8
Fig. 10. Coexpression of RBP2 does not affect the transcriptional repression
elicited by p53. The effect of coexpression of RBP2 on the transcriptional
repression function of p53 was investigated by the transient transfection-
transcription assay. Hela cells were transfected with 5 pg of the pSV2CAT
reporter, 10 pg of the p53 expression plasmid and increase amount of the
RBP2 expression plasmid, 0 pg, (lane 4), 20 pg (lane 5), and 30 pg (lane 6).
Transfection was also performed by replacing the deletion mutant 1-1201 for
the wild-type RBP2 expression plasmid (lanes 7 and 8). Cells were
transfected as controls with 5 pg of the reporter only (lane 1), 5 pg of the
reporter with and 30 pg of the RBP2 expression plasmid (lane 2), and 5 pg of
the reporter and 30 pg of the 1-1201 expression plasmid (lane 3). The
transfected cells were harvested 48 hour posttransfection and lysed by freeze-
thaw cycles. Total protein concentrations of lysates were measured with Bio-
Rad protein assay dye reagent. The endogenous acetyl transferase activity
was heat-inactivated at 65°C for 10 min. Enzyme reactions were conducted
with the same amount of total proteins from the lysates, in the presence of
0.125 pCi of D-threo-[dichloroacetyl-l-1 4 C]chloramphenicol and 1 pi of 40
mM acetyl coenzyme A. About one half of each lysate was used for the
assay. The products and unreacted substrates were extracted from the reaction
mixtures with acetyl acetate, dried, resuspended in a small volume o f acetyl
acetate, and resolved on a silica gel coated plate by thin layer
chromatography.
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GAL4-RBP2 Does Not Activate Transcription from the GAL4 Sites Containing
Promoter
The speculation that RBP2 might act as a transcriptional regulator was also primarily
approached by the transient transfection-transcription assay. The reporter
pGAL45/ElBCAT (a generous gift from Dr. Chi Van Dang, Fearon et al., 1992) for the
assay contains the CAT gene driven by the basal E1B promoter preceded with five
repeated GAL4 binding sites. The full-length and two truncated RBP2 cDNAs
corresponding to the amino acids 1064-1722 and 1200-1722 were fused to the DNA
encoding the yeast transcription factor GALA DNA-binding domain (1-147) to create
plasmids for expression of the corresponding proteins. Since the GAL4-p53 fusion
protein is able to stimulate transcription from the GALA binding sites containing
promoter (Fields and Jang, 1990; Raycroft et al., 1990), the GALA-p53 expression
plasmid was used to provide a positive control. The expression of the fusion proteins was
proved by immunohistochemistry with ABC kit (Vector) after transfection of the
corresponding cDNAs into H358 cells by electroporation as described in the following
section. The CAT activity was detected in the cell lysate prepared from H358 cells
transfected with the GALA-p53 plasmid (Fig. 11, lane 3). However, no activity was
detected from lysates of cells transfected with either of three structures derived from
RBP2 (lanes 4-6).
Cells Transfected with Wild-Type RBP2 cDNA Formed Less and Smaller Colonies
than with the Truncated Form
Cellular effect of ectopic overexpression of RBP2 was primarily explored by stable
cell transfection assay in an attempt to link the RB-, p53-, DNA-binding properties of
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1 2 3 4 5 6
Fig. 11. The GAL4-RBP2 and GAL4-RBP2 C-terminus fusion proteins can
not enhance transcription from the GAL4 binding sites containing promoter.
The ability of RBP2 and its C-terminus to enhance transcription was
investigated by the transient transfection transcription assay. H358 cells were
cotransfected with 7.5 pg of the reporter pGAL45 /E lB C A T and 15 pg of
GAL4-RBP2 (lane 4), GAL4-RBP2/1064-1722 (lane 5), or GAL4-RBP2/
1200-1722 expression plasmid (lane 6). Cells were also transfected with 7.5 pg
of the reporterand 15 pg of the GST-p53 expression plasmid (lane 3), 15 pg of
the unrelated plasmid pGEM4 (lane 1), and 15 pg of the blank vector pGALO
(lane 2). Transfected cells were harvested 60 hour posttransfection and lysed
by freese-thaw cycles. Total protein concentrations of lysates were measured
with Bio-Rad protein assay dye reagent. The endogenous acetyl transferase
activity was heat-inactivated at 65°C for 10 min. Enzyme reactions were
conducted with the same amount of total proteins from the lysates, in the
presence of 0.125 pCi of D-threo-[dichloroacetyl-l-1 4 C]chloramphenicol and 1
pi of 40 mM acetyl coenzyme A. About one third of each lysate was used for
the assay. The products and unreacted substrates were extracted from the
reaction mixtures with acetyl acetate, dried, resuspended in a small volume of
acetyl acetate, and resolved on a silica gel coated plate by thin layer
chromatography.
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RBP2 to its biological function. H358 cells were transfected by electroporation with equal
molar ratio of plasmids carrying the wild-type RBP2 cDNA and the mutant A1497-1599
cDNA with truncation of the RJB-, p53-, and DNA-binding domains. The cells were
histochemically treated with ABC kit 2 and 30 day posttransfection (Fig. 12 and Fig. 13).
Positive cells expressing RBP2 or the mutant were identified based on brown color
precipitates, the products produced from the soluble substrate 3, 3’-diaminobezidine
tetrachloride (DAB) catalyzed by horseradish peroxidase. This enzyme was biotinylated
and able to form a complex through the biotin-avidin-biotin bridge to the biotinylated
anti-rabbit IgG secondary antibody, primary rabbit anti-RBP2 serum LTE2-1, and RBP2
tripartite complex. As the product is insoluble, it was precipitated in situ immediately
upon being produced, thus the location of the brown color precipitates indicates the
cellular location of the tested protein. 2 day posttransfected cultures treated with ABC
reagents showed single positive cells with the expressed wild-type RBP2 protein
absolutely in the nuclei (Fig. 12A) and mutant protein mainly in the cytoplasm (Fig. 12B).
It was also observed that a much lower number of 2 day posttransfected cells expresses
visible amount of RBP2 than the mutant (compare Fig. 12C with 12D). 30 day
posttransfected cells formed colonies with the wild-type RBP2 protein in the nuclei (Fig.
13A) and the mutant protein in the cytoplasm (Fig. 13B) as observed in the 2 day
posttransfected cells, but the cells transfected with the wild-type RBP2 cDNA showed
smaller and 10 time less colonies than that transfected with the mutant cDNA.
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Fig. 12. Identification and localization of RBP2 and the truncated mutant in trans
fected H358 cells by immunohistochemistry. 2 day posttranfected cells were treated
with anti-RBP2 serum LTE2-land ABC reagents. The brown colored areas indicate
the locations of the expressed proteins. Cells are in A transfected with the wild type
RBP2 cDNA, magnification is x430; in B transfected with truncated RBP2 A1497-
1599, magnification is x430; in C as same as A, magnification is x860; in D as same
as B, magnification is x860.
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Fig. 13. Comparison of the potentials of colony formation in H358 cells transfect
ed with wild type RBP2 cDNA and in the same cells transfected with the truncated
mutant by immunohistochemistry. 30 days posttransfected cells were treated with
anti-RBP2 serum LTE-1 and ABC reagents. The brown colored areas indicate the
location of the expressed RBP2 protein. The magnification is x430. A formed col
ony from cells transfected with the wild type DNA in A; partial of a colony from
cells transfected with the mutant in B; partial of a colony in B; and partials of col
onies incorporated by neomycine resistance gene only in C and D , which are from
the same culture as in A and B respectively.
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D isc u ssio n
The Carboxyl-Term inal Domain 325-380 Determines the Binding Ability of Full-
Length p53 to RBP2
Mutation/deletion of P3 (372-380) and P2 (357-370) sequences in the C-terminal
domain 325-380 of p53 abrogates the abilities of both full-length p53 and its C-terminal
293-393 to interact with RBP2. This is likely due to either one of the two reasons. 1) P3
and P2 participate in controlling the conformation of p53 required for the interaction.
Mutation of these sequences impairs the domain 325-380 and also changes the
conformation of whole molecule so that the two upstream domains are hidden away. 2)
The two upstream domains are never exposed to bind p53 in the absence o f the third
protein, mutation of P3 or P2 is therefore enough to dissociate the interaction. The results
that binding by antibodies PAb421 and PAbl22 both against the P3 region diminish and
deletion o f P3 plus partial P2 abrogates the p53-RBP2 interaction indicate the possibility
that RBP2 directly interact with the P3 region of p53.
Further Investigation Is Necessary to Answer W hether Binding by RBP2 Affects the
DNA Binding and the Transcriptional Activation Function of p53
Although no stimulating effect was detected on the sequence-specific DNA binding
by p53, the possibility could not be completely eliminated that RBP2 is able to stimulate
this activity. The truncated form of RBP2 used for the assay carries only one of the two
determined p53-binding domains, and it interacts with only part of the RBP2-binding
domains o f p53 (domains 325-380 and/or 237-342), these may affect its ability to fulfil
the activation process. On the other hand, we do not know whether the activation requires
the full length of RBP2. Improvement is needed to be done when DNA-binding activity
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of p53 is tested again in future, for example, the full length of RBP2 with mutation of a
few of amino acids instead of with large region truncation can be used for the purpose. In
this case, the mutant fails to interact with double-stranded DNA but retained the relatively
intact p5 3-binding domains. Thus we may avoid bring about the undesired impact on the
investigated function ofRBP2.
Although no enhancement of the transactivity of p53 by cotransfection with RBP2
cDNA was detected in randomly growing cells, it is still possible that RBP2 stimulates
the activity of p53 under some special circumstances. It has been known that multiple
kinases such as C K -II, PKC and Cdk2 are able to activate p53 for sequence-specific DNA
binding in vitro. Combination of some of their functions may be more than enough to
fully activate p53 in actively dividing cells, which turns out to mask the stimulating effect
of R JB P 2 in the assay. Supporting this speculation is the observation that actively dividing
cells transfected with the wild-type p53 cDNA and with the mP3 cDNA (constitutively
active in DNA binding) exhibited a very similar CAT activity. Given the fact that wild-
type p53 in GO phase lacks transcriptional transactivity (Deffie et al., 1995), this effect
may be tested by synchronizing the transfected cells in the G O phase soon after
transfection. The effect on the transactivity of p53 elicited by RBP2 will be possibly
isolated from the others under this condition if there is.
DNA Binding of RBP2 Might Be Regulated by p53, RB, and pl30
Structural overlapping between the DNA-, RB-, and p53- binding domains on RBP2,
as shown in Fig. 14, suggests the possibility that p53 and RB might regulate the function
of RBP2. p53 or RB might mediate or interfere with the DNA binding by RBP2. In
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addition, RBP2 also interacts with another Rb family member p i30 (demonstrated by Dr.
Chen Li in our lab) through the sequences overlapping with the p53-, RB- and DNA-
binding domains (Fig. 14). It w ill be of great interest to know whether p53, RB, and p i 30
can enhance or prevent the DNA binding by RBP2.
The Candidate of Signal Peptide for Nuclear Localization of RBP2
The nuclear localization signal of RBP2 was found to lie within residues 1497-1599
(Fig. 14). The peptide sequence KPRKKKLKL (1538-1545) was suspected as a
candidate, which is similar to the nuclear localization signal peptides defined in SV40
large T antigen (SQHSTPPKKKRKV) (Kalderon et al., 1984), human p53
(SSSPQPKKKPL) (Addison et al., 1990), and some other nuclear proteins. Our latest
transfection experiment with a nuclear localization signals-truncated p53 cDNA
engineered to the upstream of the DNA sequence encoding KPRKKKLKL provided proof
for this speculation. This nuclear localization signal thus allowed localization of the
cytoplasm-anchored mutant p53 to the nuclei o f cells (data are not shown).
RBP2: a Growth Suppression-Related Gene?
One observation is intriguing: transfections with wild-type RBP2 cDNA and the
mutant brought about different effects on the growth of H358 tumor cells. The wild-type
cDNA transfected cells formed much less and smaller colonies 30 days after transfection
compared with the mutant cDNA transfected cells. Although the reason that RBP2 slows
down cell dividing is unknown. The failure of the mutant to enter the nucleus and to
interact with DNA, p53 and RB suggests a possible functional role o f RBP2 in either
replication or transcription, or both. The facts that hBRGl cooperates with RB to
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homeodomain
homolog.seq.
zinc finger (890-908,1181-1204)LXCXE charged
(311-340)-----------j ---------! (1373-77)(1497-1599)
■ ■ I I m I 1722
RB binding
(1352-1440,1457-1558)
■ ■
pl07 binding
(1352-1400)
TBP binding
(1457-1558)
RBP2
p53 binding
(1200-1496, 1497-1599)
pl30 binding
(1200-1457, 1497-1599)
DNA binding
(1458-1599)
nuclear loca. signal
(within 1497-1599)
E 2
F ig . 14. The potential functional domains o f RBP2. B lack
bars represent protein - or D N A -b in d in g dom ains. The
hatched bar represents the p53 binding dom ain w ith in
1 2 0 0 -1 4 9 6 o f R B P 2, w hich has not been determ ined
precisely. Num bers above the binding domains indicate
their locations in RBP2.
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suppress tumor cell growth and that RB potentiates hBrm-involved transcriptional
activation by glucocorticoid receptor (Dunaief et al., 1994; Singh et al., 1995) suggest
that cellular LXCXE domain-containing proteins are not necessarily the equivalents of
the viral oncoproteins and their functions are not necessarily growth-promotive either.
Instead, their functions may be growth suppression- or differentiation-related. However,
This observation has been so far made in only one cell line. More cell lines should be
tested in order to draw a final conclusion.
C o n c lu sio n
1. p53 physically interacts with RBP2 through three domains, 66-225, 237-342 and
325-380, and 325-380 determines the binding ability of full length p53 to RBP2.
2. RBP2 is a sequence non-specific double-stranded DNA-binding protein. The DNA-
binding domain lies within amino acid residuesl458-1599.
3. RBP2 interacts with p53 through the C-terminal two binding domains 1200-1496
and 1497-1599 with the latter overlapping with the RB-, pl30-, TBP- and DNA-binding
domains.
4. The nuclear localization signal of RBP2 is located within the amino acid sequence
1497-1599.
5. Coexpression of RBP2 affects neither transcriptional activation nor transcriptional
repression by p53 under our experimental conditions.
6. Over expression of wild type RBP2 can retard the growth of H358 tumor cells.
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M a te r ia ls a n d M e th o d s
Plasmids and Recombinant Baculoviruses
pCMVwtp53 and mutants were constructed by cloning the p53 and the mutant
cDNAs from the reformed pETwtp53 and the mutants (The BamH I site was created at
the 5’ ends of p53 and the mutant cDNAs) with BamH I sites into the same enzyme
treated pCMV-neo-Bam vector.
Plasmids expressing GST-RBP2 deletion mutants for determination of the p53-
binding domains and the DNA binding domain were cloned as shown in Table 1. The
vector for cloning was pGEX2T. pET3aRBP2C was constructed by graduate student Ling
Li.
pCMVRBP2 was a generous gift from Dr. Ed Harlow. pCMVRBP2/l-1201 was
constructed by cloning the BamH I digested cDNA fragment encoding the peptide
sequence 1-1201 of RBP2 into the same enzyme digested pCMV-neo-Bam.
pRc/CMVRBP2/D 1458-1593 was created by cloning two PCR fragments of RBP2
encoding the peptide sequences 1-1457 and 1594-1722 into the pRc/CMV (Invitrogen)
vector. Ligation was conducted between the vector digested with Not I and Apa I, the
upstream fragment with Not 1 and Xba I, and the downstream fragment with Xba I and
Apa I.
pGAL4p53, pGAL4RBP2 and mutants used in the transient transfection-transcription
assay for testing the transcriptional transactivity of RBP2 were constructed by cloning the
corresponding restriction endonucleases digested cDNA fragments into the pGALO vector
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(a generous gift from Dr. Chi Van Dang). The enzymes used in each cloning are
described in Table 2.
Table 1. Structural information of pGEX2T expression vectors carrying truncated RBP2
sequences
Plasmid ** DNA Sea. DNA source Clonine Sites
PGEXRBP2/1200-1599 3751-4953 PCR BamH I/EcoR I
pGEXRBP2/1200-1496 3751-4640 PCR BamH I/EcoR I
pGEXRBP2/l 200-1457 3751-4523 RBP2 cDNA BamH I/Bgl II
pGEXRBP2/l 200-1722* 3751-6110 RBP2 cDNA BamH I/BamH I
pGEXRBP2/1458-1722 4520-5548 PCR BamH I/EcoR I
pGEXRBP2/l 497-1722 4640-5548 PCR Bgl II/EcoR I
pGEXRBP2/1593-1722 4929-5548 PCR Bgl II/EcoR I
*pGEXRBP2/1200-1722 was constructed by graduate student Ling Li. The truncated
fragment obtained from digestion of the R JB P2 cDNA with BamH I and cloned into the
same enzyme treated pGEX2T vector.
**Numbers in the plasmid names represent the protein sequences with first and last
amino acid residues.
More plasmids kindly provided by other research groups were pSV2CAT by Dr.
Bruce H. Howard (Gorman et al., 1982) and pGAL45/EIBCAT by Dr. Chi Van Dan
(Fearon et al., 1992).
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Table 2. Cloning sites for the plasmids used for transient transfection-transcription assay
Peptide Sea. Name of Plasmid cDNA Sites Vector Sites
p53 pGAL4p53 Hinc II/Sac I BamH I(F)/Sac I
RBP2 pGAL4RBP2 Not I(F*)/Xba I BamH I(F)/Xba I
RBP2/1065-1722 pGAL4RBP2/1065-1722 EcoR I(F)/Xba I BamH I(F)/Xba I
RBP2/1200-1722 pGAL4RBP2/1200-1722 BamH I(F)/Xba I Nde I(F)/Xba I
F* represents the cohesive end filled in with Klenow large fragment enzyme.
Bacterial Strains and Insect and Mammalian Cells
Bacterial strains BL21 for expression plasmids with pET8C and pET3a backbone, and
DH5a for expression plasmids with pGEX2T backbone were described in the section of
Bacterial Strains and Cell Lines in Materials and Methods of Chapter 2.
Insect Spodoptera frugiperda (Sf9) cells used as host cells for baculoviral infection
and protein expression were described in the same section as mentioned above
Human non-small cell lung carcinoman H358 was provided by Dr. John Minna.
Cervical cancer cell line Hela was purchased from ATCC. The growth conditions for
Hela cells were as same as H358: in RPMI 1640 medium supplemented with 10% FBS,
100 units/ml penicillin G, and 100 pg/ml streptomycin at 37°C with 5% C02.
Immunoprecipitation
The manipulation was as described in the section of Immunoprecipitation in Materials
and Methods of Chapter 2.
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The Gel Electrophoretic M obility Shift Assay (EM SA)
GST-p53 and GST-truncated RBP2 fusion proteins used in the assay were purified by
basically following the protocol provided by Pharmacia for purification of GST fusion
proteins. Glutathione sepharose 4B (GS-4B) matrix (Pharmacia) was washed with 10 bed
volumes of PBS, pH 7.4, to remove the preservative. The gel bed was equilibrated in
PBS.
Bacterial cell lysates containing the proteins to be purified were prepared as described
in the section of In Vitro Phosphorylation in Materials and Methods of Chapter 2. The
lysates were rocked with GS-4B beads at 4°C for 2 hrs. The beads were separated from
lysates by brief centrifugation. The lysates were removed and the beads were rinsed with
PBS, pH 7.4, for three times. Proteins were eluted from the GS-4B matrix two to three
times with elution buffer containing 10 mM glutathione in 50 mM Tris-HCl, pH 8.0. The
eluted proteins from the same sample were collected and pooled, and concentrated in a
microcon microconcentrator (Amicon) by centrifugation at 6,000 rpm at 4°C. The
purified and concentrated proteins could be used freshly, stored at 4°C for about two
weeks, or mixed with 50% glycerol and stored at -20°C.
Manipulation of EMSA for testing DNA binding by GST-truncated RBP2 was as
same as described in the section of The Gel Electrophoretic Mobility Shift Assay in
Materials and Methods of Chapter 2 except that GST-p53 and GST-truncated RBP2 were
mixed and incubated at room temperature for half a hour before adding 3 2 P-labeled BC.
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The Transient Transfection-Transcription Assay
The experimental procedures were basically as same as described in the section of
The Transient Transfection-Transcription Assay in Materials and Methods of Chapter 2.
1. Testing the effect of cotransfection of RBP2 on the transcriptional activation
function of p53. H358 cells were used for transfection. DNA samples for transfection
contained 2.5 pg of the POSTCAT2 reporter and 2.5 pg of pCMVwtp53 combined with
increased amount (0,12.5,25, and 50 pg) of pCMVRBP2 or pCMVRBP2/l-1201. Vector
pCMV-neo-Bam was used for filling up the difference between the DNA samples. Same
total amount of proteins was used in the enzyme reaction for testing the CAT activity.
About one third of each cell lysate was used for the enzyme reaction.
2. Testing the effect of cotransfection of RBP2 on transcriptional repression function
of p53. Hela cells were grown to about 80% of confluence. The medium was changed
several hours before transfection. DNA samples for transfection contained 10 pg of the
pSV2CAT reporter and 10 pg of pCMVwtp53 combined with increased amount (0, 20,
40, and 60 pg) of pCMVRBP2 or pCMVRBP2/l-1201. Total DNA amount was adjusted
to be identical among all the samples with the pCMV-neo-Bam vector. 2.5 x 106 cells
were used for each transfection sample. Electroporation was operated under the
conditions of 250 volts, 1180 Farad, and low Q. Same total amount of proteins was used
in the enzyme reaction for testing the CAT activity. About one third of each cell lysate
was used for the enzyme reaction.
3. Testing the transactivity of GAL4-RBP2 chimeric protein from the GAL4 binding
sites containing promoter. H358 cells were used for transfection. 7.5 pg of the
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pGAL45/ElBCAT reporter combined with 15 pg of pGAL4wtp53, pGAL4-RBP2, or the
mutant DNA (pGAL4-RBP2/l065-1722, pGAL4-RBP2/l200-1722) were applied in each
transfection operation. The pGALO vector was used to fill up the difference of DNA
amount between the transfections. Same total amount of proteins was used in the enzyme
reaction for testing the CAT activity.
Immunohistochemistry
The experiment was carried out with Vectastain ABC K it from Vector Laboratories.
Transfected cells in a petri dish 48-72 hours postelectroporation were rinsed with PBS,
pH 7.4, briefly and fixed in ethanol:acetic acid (95:5) on ice for 10 min. 10 ml of PBS
were added to the cells, placed at room temperature for 10 min, and then vacuumed out.
The cells were treated in PBS with 3% bovine serum albumin (BSA), 1.5% normal horse
serum, 0.2% Triton X-100 and 0.02% NaN3 at room temperature for 30-60 min. Primary
rabbit anti-RBP2 serum LTE2-1 was added to the cells in 1% BSA and 0.02% NaN3 and
placed at 4°C overnight. The cells were rinsed next day for three times with PBS
containing 0.2% Triton X-100 at room temperature, each the solution was maintained
with cells for 10 min and then vacuumed out. Biotinylated secondary antibody against the
primary antiserum was added to the cells. The cells were incubated at 37°C for 1 hr and
then were rinsed 3 times with PBS as above. The freshly diluted (1:100 in PBS), mixed
and preincubated reagent A and B (avidin and biotinylated horse radish peroxidase
respectively, at room temperature for 30 min) were added to the cells. The cells were
incubated at 37°C for 1 hr, then rinsed 3 times with PBS as above, and incubated with 0.4
mg/ml 3,3'-diamino-benzidine (a substrate of horse radish peroxidase) and 0.015% H2O2
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in 25 mM Tris-HCl, pH 7.6. The enzyme reaction was undertaken at room temperature
for 5 min or more. The cells were rinsed with running water when the reaction was
finished and kept in water for observation and photography.
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CHAPTER 5: DISCUSSION
Previous studies have shed the light on the function of p53. The molecular
mechanisms by which p53 suppresses cell growth and induces cell death in response to
the genotoxic stress, however are still not fully understood. The research described in this
thesis has provided valuable information for further understanding the regulatory function
of the C-terminal of p53 and more candidates of p53 associated proteins.
Consisting with the previous report by Lane’s group, peptide P3 (amino acid residues
372-380) is responsible for maintenance of a latent form of p53 for DNA binding.
Peptide P5 (amino acid residues 381-393) participates in formation of the protein
conformation required by DNA binding. The same peptide also contributes to the
formation of a dimensional structure recognized by cyclin A/Cdk2 in phosphorylation.
The studies of P3 and P5 describe in more details how different peptides in the C-
terminal of p53 coordinate to accomplish the DNA-binding regulation. They have also
proved the functional significance of RB/p53 homologous sequences as viewed from p53.
Our studies show that Cyclin D preferentially interacts with C-terminal truncated p53.
Given the fact that the active form of p53 for DNA binding does not show much higher
affinity for cyclin D compared with the wild type, cyclin D might, therefore, in cells
interacts with either naturally truncated p53 or the full-length protein in the presence of
special assisting factors.
RBP2 is another cellular protein with p53 binding ability. The interaction can occur
when p53 is in the latent form for DNA binding. These two events, therefore, may
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represent two different functions of the protein since p53 is multifunctional and work
differently when it binds versus does not bind DNA.
Our further studies show the possibility that cellular proteins cyclin D and RBP2
functionally associate with p53. Failure of binding DNA by the truncated p53 and cyclin
D complex indicates a competition between cyclin D and consensus sequence for the p53
protein. Interaction of p53 with cyclin D family and its competition with RB for cyclin D
in vitro suggest that p53 might function to prevent RB from being phosphorylated in the
Gl/S boundary. Whether this interaction also interferes with the complex formation of
cyclin D and cdks needs further investigation. The interaction between p53 and RBP2
may represent one of the multiple steps on the pathway that p53 exits its growth
suppression function since over-expressed RBP2 can retard tumor cell colony formation
in our stable transfection experiment.
RBP2 has been studied in more details to understand the functional significance of its
interaction with p53. Our studies have shown that RBP2 is a non-specific double
stranded DNA binding protein. This observation is consistent with its identity of being a
nuclear protein. Inhibition of colony formation of H358 tumor cells by RBP2 provides
evidence to classify RBP2 as a growth suppressor. What is the working pathway of RBP2
and whether the suppressive function is related with its interaction with either RB or p53
will be persuaded in the future.
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Creator Wu, Kai-Jin (author)

Core Title Studies on the regulation of p53 and investigation of functional significance of interactions between p53 and its binding proteins

Degree Doctor of Philosophy

Degree Program Molecular Microbiology and Immunology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,biology, molecular,health sciences, oncology,OAI-PMH Harvest

Language English

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-189990

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Legacy Identifier 3054913.pdf

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Rights Wu, Kai-Jin

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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