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Post-translational modification of nucleolin by monoubiquitylation in human ovarian carcinoma cells

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Post-translational modification of nucleolin by monoubiquitylation in human ovarian carcinoma cells

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Content POST-TRANSLATIONAL MODIFICATION OF NUCLEOLIN BY
MONOUBIQUXTYLATION IN HUMAN OVARIAN CARCINOMA
CELLS.
Copyright 2004
by
Bongha Shin
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERISTY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHGBIOLOGY)
December 2004
Bongha Shin
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UMI Number: 3155478
Copyright 2004 by
Shin, Bongha
All rights reserved.
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DEDICATION
This work is dedicated to my father, mother, brother, wife and son who always bring
their everlasting love and joy into my life.
But without God’s love and grace this work could not be done.
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ACKNOWLEDGEMENTS
I really thank Dr. Louis Dubeau who makes me as one qualified scientist by giving
unfathomable intellectual and emotional supports. It is my utmost pleasure to have
Dr. Michael Stallcup, Dr. Robert Stellwagen, and Dr. Randall Widelitz as my
committee member, and I appreciate their guide. I also want to give my special thank
to Ms. Lisa Doumak who helped me with all her heart.
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TABLE OF CONTENTS
DEDICATION I I
ACKNOWLEDGEMENTS H i
LIST OF FIGURES vi
ABSTRACT vii
CHAPTER I
INTRODUCTION................... 1
Role of ubiquitylation on the regulation o f protein degradation and function 1
Intracellular proteolytic pathways. .................... 1
Components o f the ubiquitylation machinery. ...... 4
Monoubiquitylation regulates the localization and function of target protein
8
1) H istones.................. 8
2) Plasma membrane receptors ....... 9
3) Retroviral budding ......... 10
4) Fanconi Anem ia.................. 1 1
Determinants o f mono- versus poly-ubiquitylation o f proteins ............12
Ubiquitin in human diseases ...................... 13
SUMO: modulator or competitor for ubiquitylation? ................... 14
Metastasis and its association with Galectins ............................ ....17
Protease mediated local invasion o f tumor cells ........... 19
Rate limiting factor for metastasis: The adhesion of circulating cancer cells to
the wall of endothelium .................... 20
Lectins: selectins in lymphocyte homing .......................... 22
Galectins: specific [3-galaetoside recognizing lectins ...................... 23
Galectins modulate cell-cell and cell-matrix adhesion ...................... 25
Galectins modulate cell proliferation and apoptosis ......... ..26
Galectin-3 in inflammation ........... .....28
Galectin-3 in tumorigenesis and metastasis ............. .....29
HYPOTHESIS ........ ...31
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CHAPTER H
POST TRANSLATIONAL MODIFICATION OF NUCLEOLIN
BY MONOUBIQUITYLATION IN HUMAN OVARIAN CARCINOMA CELLS
Abstract ........................................................ .33
Introduction .3 5
Materials and M ethods................ 41
Results ............................. 46
Discussion ............................................................. .63
CHAPTER H I
POST-TRANSLATIONAL MODIFICATION OF GRP78 BY SUMOYLATION
IN HUMAN OVARIAN CARCINOMA CELLS
Introduction................ 69
Materials and methods.............................. 72
Result and discussion............... 75
CHAPTERVI
CONCLUSIONS AND FUTURE DIRECTIONS
Conclusions and Future Directions ............................. ..78
REFERENCES ............................ 83
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LIST OF FIGURES
Figure 1. Differential ubiquitylation o f nuclear proteins in an in vitro longitudinal
model for ovarian cancer progression..................... 47
Figure 2. Identification of the 105 kDa ubiquitylated fragment as nucleolin 49
Figure 3. Differential expression o f monoubiquitylated nucleolin in various human
ovarian cell lines.................... ...51
Figure 4. Association between nucleolin monoubiquitylation and phosphorylation
in ovarian cell lin es.............................. 56
Figure 5. Expression and purification o f GST-Nucleolin fusion protein ....59
Figure 6. Monoubiquitylation may induce the conformational change of nucleolin.
61
Figure 7. Model for the role of monoubiquitylation and phosphorylation in
regulating intracellular localization o f nucleolin. ........................ 68
Figure 8. Differential sumoylation o f GRP78 in ML-10 and MCV-50 cells. ......76
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Vll
ABSTRACT
Proteins with polyubiquitin chains, in which lysine 48 of ubiquitin binds to
the C- terminal glycine o f the next ubiquitin molecule, are degraded by the
26S proteasome complex. Monoubiquitylation, single ubiquitin conjugation without
polymer chain formation, does not result in protein degradation but it regulates the
location and function of its conjugating proteins. Here we report that Nucleolin, a
major eukaryotic phosphoprotein responsible for ribosome biogenesis and
maturation, is monoubiquitylated in various human ovarian carcinoma cell lines
and that the majority o f monoubiquitylated nucleolins exist in the nuclear
compartment of cells.
Cytoplasmic and nuclear proteins from ovarian carcinoma cells were
further separated into phosphorylated and non-phosphorylated proteins and
immunoprecipitated with an antibody against ubiquitin. The only form of
monoubiquitylated nucleolin found in the cytoplasm was phosphorylated while
phosphorylated and non-phosphorylated forms were both present in the nucleus,
with a predominance o f the non-phosphorylated form. This monoubiquitylation
may cooperate with phosphorylation to control its cellular localization.
SUMO (small ubiquitin modifier) also post-translationally modifies target
proteins. It does not mediate protein degradation, but rather modulates the
localization and function of substrates.
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Glucose-regulated protein 78 (GRP78), an essential component for protein
processing and folding in ER, cell homeostasis and regulating apoptosis, is
sumoylated in our ovarian cystadnoma cell line.
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CHAPTER I
1
INTRODUCTION
Post-translational modifications play pivotal roles in modulating the function,
degradation, and localization o f intracellular proteins. Through such modifications, a
single gene can generate as many as 50 different protein species, often producing
means of regulating protein activity. This thesis addresses the question of how post-
translational modifications such as ubiquitylation and glycosylation may affect
ovarian cancer development and spread. The fields of ubiquitylation and of
metastasis-associated glycosylation, the latter focusing on the galectin protein
family, are reviewed in this chapter in order to provide background information to
my thesis work.
Role of ubiquitylation on the regulation of protein degradation and function
Intracellular proteolytic pathways
The level o f intracellular proteins is not only determined by rate of protein
synthesis, but also by protein degradation. The half-life o f intracellular proteins
ranges from minutes to a few weeks.
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In eukaryotic cells, two major pathways, the lysosomal proteolysis pathway and the
cytoplasmic ubiquitin-proteasome pathway, regulate protein degradation. It is
believed that the lysosomal proteolysis pathway degrades most long-lived proteins -
proteins with more than five hours lifetime - while the ubiquitin-proteasome pathway
degrades short-lived regulatory proteins (1).
Lysosomes are membrane-enclosed organelles that contain many proteases
and digestive enzymes. This containment of such enzymes within lysosomes
prevents uncontrolled degradation of cellular contents. Therefore, in order to be
degraded by lysosomal proteolysis, proteins have to be taken up by lysosomes.
Lysosome mediated uptake of cellular proteins can be done by endocytosis, macro-
or micro- autophagy, vacuolar import, and direct transport (2).
Unlike lysosomal proteolysis, which generally functions as non-selective
protein degradation in cells, the ubiquitin-proteasome pathway is responsible for
selective protein degradation. Ubiquitin is a highly conserved 76 amino-acid
eukaryotic protein that can be conjugated to other proteins or to itself through
isopeptide bonds between the C-temiinai Gly76 o f ubiquitin and a Lys residue in a
target protein. Proteins with polyubiquitin chains, in which lysine 48 of ubiquitin
binds to the C-terminal glycine of the next ubiquitin molecule, are degraded by the
ATP dependent 26S proteasome complex (3).
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The signals that target proteins for this ubiquitin-proteosome degradation are
still incompletely understood. Although mechanisms of recognition through the N-
terminal residues of substrates called N-end rule pathway (3) has been studied
extensively, this is not a major signal that targets intracellular proteins for the
proteasomal degradation. Proteasome is composed of two sub-complexes, a core
catalytic 20S particle and a regulatory 19S particle that caps a core particle. The 20S
complex is barrel-shaped and consists o f four stacked rings, two identical outer a
rings and two identical inner p rings. The a and p rings are composed each of seven
distinct subunits, giving the complex the general structure of a (l -7)P( 1 -7)P 1 -7)a (1-
7). The catalytic sites are localized in p subunits (4). 19S particle recognizes poly-
ubiquitylated proteins and unfolds the polypeptide chains so they can be inserted into
the 20S proteolytic chamber (5). As mentioned above, only proteins with
polyubiquitin chains bound at Lys 48 are targeted for proteosomal degradation.
Although the functions of polyubiquitin chains connected through Lys 11 and 29
remain to be discovered, polyubiquitin chain linked by Lys 63 is involved in
important regulatory events, such as DNA repair, translation, IkB kinase activation,
and endocytosis and vesicle transportation (6).
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Components of the ubiquitylation machinery
Ubiquitylation is a process involving a sequence o f multiple enzymatic
reactions. Ubiquitin is activated by the ATP dependent ubiquitin-activating enzyme
(E l), followed by interaction with the ubiquitin-conjugating enzyme (E2). In some
cases, for example, histone ubiquitylation, the activated ubiquitin molecule is
transferred from E2 to the target protein, but in most cases conjugation o f activated
ubiquitin to target proteins is catalysed by ubiquitin ligase (E3) (7). Therefore, it is
likely that there are numerous E3 ligases even though relatively few such enzymes
have been isolated so far.
Most E3 ligases fall into two distinct families, the HECT domain family and
the RING finger domain family. The discovery o f HECT (Homologous to E6-AP
Carboxyl Terminus) domain came from observations with human papillomaviral
protein E6 and its cellular partner E6-AP. E6 plays a role as an adaptor between
E6-AP and p53, catalysing E6-AP mediated ubiquitylation o f p53 (8, 9).
Many HECT domain E3s, but not E6-AP, also have a WW domain, a protein module
that binds proline-rich or proline-containing ligands, in their N-terminus, which is
responsible for generating a hydrophobic pocket rich in proline, phosphoserine, and
phophotheronine (10).
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In addition, most WW domains o f HECT E3s have a C2 domain in their N-
terminus that translocalizes the E3 to the plasma membrane in response to
intracellular calcium increase (11). Nedd4 (neuronal precursor cell developmentally
down-regulated) has HECT, WW and C2 domains. Nedd4 and its homolog
established a family of Nedd4/Nedd4-like E3. Nedd4 ubiquitylates subunits of the
epithelial sodium channel, resulting in the down-regulation of active sodium
channels (12).
RING finger E3s are defined by their unique eight metal-binding residues
that interleave two zinc ions. They are further divided into single-subunit E3s and
multi-subunit E3s. Single-subunit E3s, such as Mdm2 (Murine double minute 2) and
the IAPs (inhibitors of apoptosis), have the target recognition motif and the RING
finger domain on the same polypeptide chain. Mdm2 is over-expressed in many
human soft tissue tumors and an elevated Mdm2 level in cancers has been associated
with poor prognosis (13). Mdm2 can ubiquitylate p53 and itself, inducing
cytoplasmic localization of ubiquitylated p53 as well as targeting ubiquitylated p53
and Mdm2 to proteasomes (14). The ubiquitin ligase activity o f Mdm2 can be
inhibited by binding o f the pRB (retinoblastoma protein) or the Mdm2 homologue
Mdmx or P19A R F(1 5 ,16,17). Members of IAP family are distinguished by the
presence o f one or more BIR (baculovims inhibitor o f apoptosis protein repeat)
motifs, which inhibit the activity of many caspases. Many IAPs, such as XIAP,
cIAPl and cIAP2, have a RING finger domain at their C-terminus.
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Upon apoptotic stimuli, XIAP and cl API are autoubiquitylated through their RING
finger domain (18,19). The sites o f substrate interaction for single-subunit RING
proteins may be highly varied. For example, the IAP’s BIR domain probably
facilitates binding of some substrates and Mdm2 interacts with p53 through its N-
terminal domain, whereas the RING domain is located at its C-terminus (15).
SCF (Skp 1 /Cullin-1 /F-box protein) complexes, VHL-CBC (von Hippel
Lindau-Cullin-2/elongin B/elongin C) and APC (anaphase promoting complex) are
known as multi-subunit E3s because they are generally composed of small
noncanonical RING finger proteins, Cullins, adaptor proteins, E2s and other
substrate recognizing proteins. As RING finger proteins, Rbxl (also called R od and
Hrtl) in SCF and VHL-CBC and Apcl 1 in APC are identified. Cdc53 (cullin-1) in
SCF, cullin-2 in VHL-CBC and Apc2 in APC compose Cullin family of proteins.
Known adaptor proteins consist o f Skpl in SCF, elongin B/C in VHL-CBC and
multiple APC subunits in APC. Other substrate recognizing proteins are F-box
protein in SCF, VHL in VHL-CBC, and Cdc20/Hctl in APC (20,21, 22). Rbx 1 is
known as an essential component of SCF or VHL-CBC complexes since it is
sufficient to mediate ubiquitylation in vitro (2 3 ,24).
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SCF complexes catalyse the ubiquitylation of diverse phosphoproteins, such
as G1 cyclins, Cdk inhibitors, and transcription factors, throughout the cell cycle
while APC, which is active from the beginning o f anaphase to the end o f mitosis and
during the period o f G1 phase, catalyses the phosphorylation-dependent
ubiquitylation of anaphase inhibitors, mitotic cyclins, and components o f the mitotic
spindle (21). VHL-CBC ubiquitylates HIFloc (hypoxia inducible transcription factor
la ) to induce VEGF (vascular endothelial growth factor) expression.
Architecturally, SCF complexes are closely related to the VHL-CBC complex. VHL
mutants that fail to assemble with CBC core complex were associated with the
malignancies of VHL-associated tumors (25,26,27).
It has been proposed that E3s can selectively recognize phosphorylated
proteins for poly-ubiquitylation. Proteins containing PEST elements, rich in Pro,
Glu, Ser, and Thr residues, can be rapidly degraded by the ubiquitin-proteasome
pathway, and actually multiple phosphorylations within the PEST elements are
required for the degradation of the yeast G1 cyclins (28). The Ik^cc o f the NFkB
requires phosphorylation at Ser 32 and 36 prior to the ubiquitylation (29). However,
protein phosphorylation can prevent ubiquitin-proteasome degradation in other cases.
Phosphorylation of the c-mos, c-fos and c-jun proto-oncogenes by MAP kinases
suppress their ubiquitylation and degradation (30).
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Monoubiquitylation regulates the localization and function o f target protein.
In contrast to polyubiquitylation, monoubiquitylation, the conjugation of a
single ubiquitin molecule to one or more Lys residues in a target protein, is a
reversible modification that is not associated with 26S proteasome-mediated
proteolysis. Ubiquitin has several Lys residues that can form polyubiquitin chains
linked through Lys 11,29, 48 and 63 in vivo (31, 32).
Several proteins are known to be monoubiquitylated in vivo or in vitro, and
recent studies showed that the monoubiquitylation regulates the location and
functional activity of those proteins. The known functional modifications of
monoubiquitylated proteins are described in the following paragraphs.
1) Histones
Eukaryotic chromatin structure is modulated by the post-translational
modification of histones. Although it has been well established that acetylation of
histone N-teraiina! directly links to chromatin remodelling and transcriptional
regulation, the functional significance o f other histone modifications remains to be
elucidated. Monoubiquitylation of histones have been associated with activation of
gene expression. C-terminal o f histones H2A, H2B and H3 are mono-ubiquitylated
and these monoubiquitylated histones are stable in vivo.
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Two ubiquitin-conjugating enzymes (E2), Rad6/Ubc2 and Cdc34/Ubc3, can mono-
ubiquitylate histones in vitro without E3 (33). In yeast, mutations o f the conserved
ubiquitylation sites o f H2A and H2B affect mitotic and meiotic cell divisions. In
addition, rad6 mutants do not have the mono-ubiquitylated H2A and H2B (34, 35). It
is suggested that the mono-ubiquitylation of H2A and H2B may alter the
conformation of histones to open chromatin structure. In Drosophila melanogaster
embryo, TATA box binding protein (TBP) associated factor (TAF 250)
monoubiquitylates histone H I. Abolishing the ubiquitin enzyme activity of TAF 250
by point mutations reduced the cellular level o f mono-ubiquitylated HI and
downregulated mesoderm-determining genes, twist and snail, targeted by the
maternal activator Dorsal. TAF 250 seems to have both El and E2 activities since it
has domains that are homologous to El and E2 (36,37).
2) Plasma membrane receptors
The receptor tyrosine kinase (RTK) signalling can be terminated by endo-
cytosis followed by lysosomal degradation (38). Ligand-induced mono
ubiquitylation of plasma membrane receptors, including many RTKs, has been
suggested as a major mechanism that regulates their internalization and endocytosis-
iysosomal degradation (39,40). In yeast, the cytoplasmic domain of the G protein
coupled receptor Ste2p is monoubiquitylated, resulting in the removal of Ste2p
through the yeast vacuole, an equivalent of the mammalian lysosome (41).
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Chimeric interleukin-2 receptor a chain and epidermal growth factor receptor
(EGFR), which have monoubiquitylated cytoplasmic domains, are down regulated
by lysosomal degradation in mammalian cells (42). Thus, the monoubiquitylation
seems to be associated with endocytosis-mediated protein degradation. However,
some evidence indicates that monoubiquitylation is not always required for receptor
endocytosis. The proto-oncogene Cbl can monoubiquitylate and down-regulate
EGFR, platelet derived growth factor (PDGF) receptor and colony-stimulating factor
(CSF)-l receptor the conditions where endocytosis was impaired (43,44). Also,
inactive EGFRs are internalized into early endosome compartment independent of
monoubiquitylation.
3) Retroviral budding
Large amounts of free ubiquitin molecules were found in several retro
viruses including avian retroviruses, human immunodeficiency virus (HTV)-l, simian
immuno-deficiency virus (SIV), and murine leukaemia virus (MLV) infected cells
(45). It was later shown that retroviral budding depends on the monoubiquitylation of
the retroviral Gag protein (46). Gag proteins are synthesized on free ribosomes and
are subsequently attached to the plasma membrane by their N-terminal membrane
binding (M) domains. Then, Gag proteins are tightly packed together by their
interaction (I) domains in order to form complexes.
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The M and I domains initiate buds formation on the surface of the infected cells, but
they are not released until the late (L) domain is present. The L domain has a proline-
rich sequence that can interact with Nedd4 ubiquitin ligase (47). Budding of
retrovirus depends on Nedd 4-mediated monoubiquitylation of L domain (48). This
is supported by the fact that low levels o f free ubiquitin in infected cells affected
virus budding, while fusion of ubiquitin to viral Gag protein could overcome this
problem (49).
4) Fanconi Anemia
Interference with a normal monoubiquitylation pathway has been associated
with the pathogenesis o f Fanconi Anemia (FA), an autosomal-recessive genetic
syndrome characterized by chromosome instability, hypersensitivity to DNA cross-
linking agents, and increased predisposition to cancers such as leukemias and
squamous cell carcinomas o f head and neck as well as gynaecologic tumors (50).
The products o f seven cloned FA genes (A, C, D l, D2, E, F, and G) and BRCA
proteins constitute FA/BRCA pathway. In response to DNA damage, the FA protein
complex composed o f five FA proteins (FANCA, FANCC, FANCE, FANCF and
FANCG) catalyses the monoubiquitylation o f FANCD2 protein. The mono
ubiquitylated FANCD2 is targeted to chromatin-associated nuclear foci where it
assembles with BRCA1, BARD, BRCA2, Rad 51 and MRE1 l/Rad5Q/NBSl
(M/R/N).
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This is required for the homologous recombination repair o f damaged DNA (51).
Recently, the breast and ovarian cancer susceptibility gene (BRCA2) was identified
as the FANCD1 gene (52). Patients with FA have lost at least one of eight different
genes coding FA proteins, resulting in the impairment o f mono-ubiquitylation of
FANCD2 (53).
Determinants of mono- versus polvubiquitvlation of proteins
How the ubiquitin system decides whether a given protein target is to be
mono- or polyubiquitylated is a major unanswered question. One proposal is that
different subsets of ubiquitin ligases may have specificity for those modifications.
This hypothesis is based on the observation that Mdm2 only mediated the
monoubiquitylation o f p53, and that the polyubiquitylation of p53 was subsequently
completed by p300 (54). Another possibility is that the same ubiquitin ligase may
mediate either mono- or polyubiquitylation depending on the nature of target
proteins or their intracellular localization. Cbl polyubiquitylates cytoplasmic
proteins, such as Sprouty, Src and Abl, but it can monoubiquitylate RTKs and Cbl
associated adaptor protein CEN85 in the endosome-lysosome pathway (55, 56).
Also, target proteins may determine the type of ubiquitylation. The ubiquitin
interacting motif (UIM) and Cue-1 homologous domain (CUE) are believed to
mediate mono-ubiquitylation o f proteins for endocytosis.
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One assumption is that proteins with UIM or CUE may transiently bind to E3 and
dissociate after binding of one ubiquitin molecule (57). Another hypothesis is that
the binding o f UIM or CUE domains to ubiquitin sterically hinders the formation of
a polyubiquitin chain because Lys 48 o f ubiquitin may be masked by the UIM/CUE
ubiquitin complex (58, 59). Ubiquitin can be removed from this complex by a de-
ubiquitylating (DUB) enzyme (60). Thus, it is possible that a balance between E3
and DUB may determine whether a protein is to mono- or polyubiquitylated.
Ubiquitin in human diseases
Malfunction of the ubiquitylation system has been observed in various human
diseases, such as cancer, cystic fibrosis, hypertension, viral infection and
neurological disorders. In uterine cervical carcinoma caused by high-risk strains o f
the human papilloma virus, the p53 tumor suppressor is degraded by E6-AP
mediated polyubiquitylation (8). Under normal conditions, P-catenin undergoes rapid
ubiquitylation either by a phosphorylation-dependent E3 complex comprised of
glycogen synthase kinase (GSK3P), casein kinase I a (CKIa), APC tumor
suppressor gene and axin, or by a phosphorylation-independent E3 complex
consisting of Skpl, APC, SIP and Siahl (61, 62). In colorectal cancer, hepatocellular
carcinomas, and melanomas, p-catenin is upregulated as well as stabilized due to the
abrogation o f ubiquitylation-mediated degradation.
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The breast and ovarian cancer susceptibility gene BRCA1 contains a RING
finger domain in its N-terminal region, a nuclear localization signal region, protein
interaction domains, and tandem BRCT domains in its C-terminal region. The N-
terminal RING finger domain ofBRCAl interacts with another homologous RING
finger protein, BARD1 (63). BRCA1-BARD1 heterodimer has E3 enzyme activity
that may either mono- or polyubiquitylate various target proteins such as FANCD2,
histones, SWI/SNF related protein, and RNA polymerase II in vitro, although this
remains to be shown in vivo (64,65). In addition, BRCA1-BARD1 complex can
interact with DNA repair molecules, p53 and apoptosis related ZBRK (66). The
mutations in N-terminal RING finger domain ofBRCAl are closely associated with
familial breast and ovarian cancer predisposition (67, 68).
SUMO: modulator or competitor for ubiquitylation?
Several proteins with sequence similarity to ubiquitin have been discovered.
These ubiquitin like proteins are divided into two classes: ubiquitin-like modifier
(UBL) and ubiquitin domain protein (UDP). UBLs are also functionally similar to
ubiquitin, and small ubiquitin related modifier (SUMO), NeddS (Rubl), Apg 8 and
Apgl2 belong to this family (2). UDPs, such as parkin, Rad23 and DSK2, have
sequence homology to ubiquitin, but they are not conjugated to target proteins (69).
SUMO (also called sentrin, GMP1, PIC-1) and ubiquitin share only 18 % sequence
homology, but their three-dimensional structures are almost identical (70, 71).
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Sumoylation is mechanistically similar to ubiquitylation, but enzymes involved in
sumoylation are distinct from enzymes catalyzing ubiquitylation. SUMO-specific
El is a heterodimer composed of AOS! and UBA2 and has sequence homology to
UBA1 ubiquitin-specific El (72, 73). SUMO has a single E2 called UBC9, but it can
efficiently distinguish between SUMO and ubiquitin molecules. The surface of
UBC9 is positively charged, which is complementary to negatively charged SUMO
(74, 75). Ubiquitin cannot bind to UBC9 due to its positive charge. Although SUMO
E3 has not been identified yet, it is believed that UBC9 alone can recognize target
proteins. In contrast to ubiquitin, SUMO cannot make poly-SUMO chains because it
does not have any branch-point Lys (29, 48, 63) residues.
The functions of sumoylation are divided into two categories. First, it can be
an address tag for protein targeting. Ran GTPase activating protein (RanGAPl) is a
cytoplasmic protein and a key regulator of the Ras-like GTPase (Ran) that controls
nucleo-cytoplasmic transport (76). Only the sumoylated RanGAPl can stably
interact with Ran binding protein 2 (Ran BP2) at the cytoplasmic face of the nuclear
pore complex (77, 78). This indicates that sumoylation either targets RanGAPl to
the nuclear pore complex or stabilizes RanGAP 1 -RanBP2 complex. PML
(ProMyelocytic Leukemia) is a RING finger protein and a component o f PML
nuclear bodies in which sumoylated proteins are concentrated (79). Sumoylation of
PML relocalizes transcriptional co-repressor Daxx to nuclear bodies, which
subsequently removes Daxx mediated repression o f genes.
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PML acts as a transcriptional co-activator in association with p53 (80, 81).
Sumoylated PML directs p53 to nuclear bodies, which leads to activation of
apoptosis and acetylation of p53 (80). HEPK2 is a Ser/Thr kinase that interacts with
homeo-domain transcription factors and acts as a transcriptional co-repressor.
Sumoylated HIPK2 is only found in nuclear bodies (82).
The second proposed function o f sumoylation is that it can act as an inhibitor
of ubiquitylation. Upon tumor necrosis factor (TNF)-mediated stimulation, IkB o c . is
phosphorylated and ubiquitylated for proteasomal degradation, which can activate
NFicB-mediated transcription. SUMO can compete with ubiquitin on the same Lys
residue of IicBa (83). As is true of E3 ubiquitin ligase for p53, the ubiquitin-
proteasome pathway also regulates Mdm2. It has been reported that SUMO can
interfere with the ubiquitylation of Mdm2 by competing for the same Lys residue.
In response to DNA damage, Mdm2 may be de-sumoylated in order to stabilize p53
(84). These observations suggest that a better understanding of the relationship
between ubiquitylation and sumoylation will have an important impact on our
understanding of post-translational regulation of protein activity.
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M etastasis and its association with Galeetins
Cancer arises not only from the accumulation o f genetic changes, but also
from alterations in gene expression and post-translational modifications, deregulating
cells from homeostatic growth control. When most malignant solid tumors are
diagnosed, there is a strong chance that some o f these cancer cells have broken off
from the initial tumor and are on their way to distant sites o f the body. This is called
metastasis, the most life-threatening aspect o f cancer because the patients can no
longer be cured by local therapy alone and eventually succumb to injuries and
complications caused by cancer dissemination and cytotoxic therapies.
The initial phase of tumor cell evasion from its primary site of origin needs
phenotypical changes such as loss of cell-to-cell adhesion and cytoskeletal
rearrangements (85). This process allows epithelial cells to acquire motile features,
which are spatially and temporally regulated. Cell-to-cell interactions are mediated
by cell adhesion molecules (CAMs), which act as both receptors on one cell and
ligands for another cell. As one of the CAMs, cadherins play an essential role in the
initiation and stabilization ofhomotypic binding o f cells to each other. The
cytoplasmic domains of cadherin bind to either ($- or y-catenin protein, which in
turn bind a - catenin, linked to the actin cytoskeleton. Tumor cells with mutated or
down-regulated a - catenin showed increased metastatic potential (86, 87).
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18
While the constitutive activation of receptor tyrosine kinases (RTKs) and
their downstream effectors such as mitogen activated protein kinase (MAPK) or
phosphatidyl-inositol (PI3) kinase increase cellular proliferation rate, cytokines such
as TGF-P, which stimulates cell cycle arrest and apoptosis, induce and maintain
phenotypical changes o f epithelia in association with active Has signaling (88, 89,
90). In addition, changes in extra-cellular matrix (ECM) composition and activation
o f ECM-degrading proteases play key roles in initiating metastasis.
The ECM consisting of secreted proteins and polysaccharides fills the space
between cells and binds cells together through cell surface receptors called integrins.
Integrin- mediated clustering between the ECM and the intracellular actin
cytoskeleton can regulate focal adhesion kinase (FAK) signaling pathways and
growth factor signaling pathways such as Ras-MAPKinase, protein kinase C, and PI3
kinase pathways (91, 92). Basement membranes, a specialized type of ECM, serve as
a support for epithelial cells and act as a sieve for transport o f nutrients and cellular
metabolic products, and for preventing free passage o f cells except lymphocytes. The
loose connective tissue beneath epithelial cell layer and basement membranes
consists predominantly of ECM in which fibroblast and capillaries are distributed.
Cross talk between epithelial cells and ECM components creates a
microenvironment for proliferation and cellular motility (93).
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Protease mediated local invasion o f tumor cells
19
The basement membrane barrier can be breeched by a variety of proteases
released from tumor cells that also can degrade ECM components in order to allow
tumor cells to invade tissue barriers as well as the walls of blood or lymphatic
vessels. All four categories of proteases-serine, cysteine, aspartic protease and
metallo-protease-have been implicated in the invasive process.
The serine protease urokinase-type plasminogen activator (uPA) and its
receptor (uPAR), which can activate plasmin, have attracted considerable attention in
the context o f tumor cell invasion. The active form o f plasmin also activates matrix
metailo-proteinases (MMPs). When inactive pro-uPA binds to uPAR, it is cleaved to
active uPA by plasmin. In turn, uPA activates plasminogen by cleaving it to form
plasmin, so a positive-feedback loop between uPA and plasmin can be formed (94).
uPA also cleaves fibronectin and its own inhibitor, plasminogen activator inhibitor-1,
in a plasminogen independent manner (95). In addition to its activation of uPA,
uPAR is thought to be involved in many protein-protein interactions related to cell
adhesion and signal transduction. It is associated with several members o f the
integrin family and involved in the initiation of mitogen activated protein kinase -
extracellular signal regulated kinase (MEK-ERK) and Janus kinase - signal
transducer and activator of transcription (JAK-STAT) pathways (96). Thus, uPA and
uPAR not only generate active plasmin, but also regulate cell adhesion and
migration.
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20
MMPs can hydrolyse a diverse spectrum o f extracellular proteins.
Although there is functional overlap between subclasses o f MMPs, they are
classified into four subclasses on the basis of their substrate specificity and sequence
homology. They are collagenases (MMP1, MMP8 and MMP13), stromelysin
(MMP3, MMP10, MMP11, MMP7, and MMP26), gelatinases (MMP2 and MMP9)
and membrane type MMPs (MMP14, MMP15, MMP16, MMP17, MMP24 and
MMP25).
MMP activity is regulated by gene expression, plasmin activation, and
inhibition by their specific tissue inhibitors (TTMPs) (97,98). Many in vivo studies
from MMP-deficient mice showed that increased expression o f MMPs is closely
associated with tumor invasiveness, angiogenesis and metastasis (99). Cathepsin B, a
cysteine protease of the papain family, can degrade components o f the ECM and
activate MMPs. It is responsible for the shifting the balance to MMPs by inactivating
TIMPs(lQO).
Rate limiting factor for metastasis: The adhesion of circulating cancer cells to the
wall of endothelium
Invasive cancer cells reach circulatory conduits such as lymphatic or blood
channels in order to disseminate into distant tissues or organs. The local invasion
process is much more efficient than actual metastasis since millions of cells shed into
circulation daily, but only a very small fraction of these cancer cells can take root in
another part of the body.
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21
Therefore, one o f the rate limiting steps in metastasis is the adhesion of circulating
cancer cells to the micro-vascular endothelium (101). The neoplastic processes often
involve the subversion o f normal cellular processes for the benefit o f the tumor.
Lymphocyte homing and recirculation system is characterized by the controlled
expression o f adhesion molecules and receptors on circulating cells and the proper
expression o f the corresponding ligands on endothelial cells. This mechanism seems
to be directly associated with the organ specific homing o f cancer cells. Three major
types of metastatic homing mechanisms have been proposed.
The first is that tumor cells may bind any organs in body but selectively grow
only in the organ that have the optimal environment with appropriate growth factors
and ECM (102). The second mechanism is selective chemotaxis whereby circulating
tumor cells are attracted to the specific organs because o f the production of soluble
chemo-attractants (103). The third mechanism proposes that circulating tumor cells
may selectively bind to the over-expressed specific cell adhesion molecules on the
endothelial surface (104). Inducible endothelial cell adhesion molecules such as
lectins establish a decisive contact between circulating tumor cells and microvascular
endothelial cell (105). Therefore, investigating the functions of cell adhesion
molecules may enhance our understanding of the mechanisms of metastasis and may
lead us to find therapeutic target for inhibiting tumor spread. The following
paragraphs especially focused on a specific family of carbohydrate binding proteins
called galectins.
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Lectins: selectins in lymphocyte homing
22
Lectins are carbohydrate-binding proteins that recognize specific oligo
saccharide structures on glycoproteins and glycolipids. Thus, lectin binding can lead
to cross-linking and aggregation o f specific glycoprotein and glycolipid receptors on
the surface o f cells, resulting in signal transduction effects such as mitogenesis,
differentiation and apoptosis.
Most animal lectins can be categorized into two classes: calcium dependent
C-type lectin, such as selectins and pentraxins, and calcium independent S-type
lectins including galectin (106,107,108,109).
The members o f selectins were identified on activated endothelial cells (E-
selectin), lymphocytes (L-selectin) and activated platelets (P-selectin). The selectins
possess rather similar carbohydrate specificities and show a considerable redundancy
o f function. They are type I trans-membrane proteins and their extracellular region is
composed of a N-terminal calcium dependent lectin domain (CRD), an epidermal
growth factor like motif and short consensus repeat (SCR) units, which are
homologous to complement-binding proteins (110, 111). The peculiar molecular
arrangement with 6 SCR segments in E-selectin and 9 SCR segments in P-seiectin
leads to the projection o f the CRD into the bloodstream. Ligands with sialylated and
fucosylated lactosamines are specific for selectin binding. In inflammatory
processes, selectins and their ligands initiate the attachment o f lymphocytes along
the vessel wall.
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23
Soon after this attachment, the inflammatory cytokines derived from the site of
infection provoke signals in both lymphocytes and endothelial cells, which result in
an increased affinity of lymphocyte integrins to their intercellular adhesion
molecules (ICAMs) on the endothelial lining (112).
Galectins: specific B-galactoside recognizing lectins
The galectins are a family o f proteins defined by homologous carbohydrate
recognition domains that recognize and bind to p-galactoside moieties on glyco
proteins and glycolipids (113). Fourteen different members of the galectin family
have been identified so far, and presently four mammalian galectins, galectin-1,2, 3
and 4, have been well characterized. Galectins 5 through 14 are included in this
family on the basis of the presence o f a carbohydrate recognition domain (CRD)
known to be required for carbohydrate binding (108,114,115,116). The general
designation of the genes encoding galectins is LGALS (lectin, galactoside binding,
soluble), and gene numbers are kept in a manner consistent with the numbering of
the proteins. Galectin-1 and galectin-2 are homodimers formed by extended P-sheet
interactions across the two-monomer subunits. Since each dimer has two galactoside
binding sites, galectin-1 can mediate either intra-molecular or inter-molecular cross-
linking (117). Gaiectin-3 is a monomer containing a C -terminal CRD, a N-terminal
domain and an intervening RNA binding Pro, Giy, Tyr-rich domain.
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24
It can form muitimers when binding to surfaces that contain glycoprotein ligands.
The N-terminal tail is required for this self-association (108).
Galectins are expressed in several tissues and are found on the cell surface,
within the extracellular matrix, in cytoplasm and nucleus o f cells. Although galectin
lacks a signal peptide, it is actively released from cells by partially understood non-
classical secretory pathways (114). Galectin-1 is synthesized on free ribosomes and
later acetylated at the N-terminus, which is typical for cytosolic, non-secreted
proteins. Actually, galectin-1 was predominantly found in the cytoplasm even though
it is also present in nucleus. DNA methylation or IL-1, TGF-P and INF-y can control
the expression of galectin-1 (118,119).
Galectin-3 is localized in both the nucleus and the cytoplasm of various cell
types and its distribution depends on the proliferation state o f the cells. Galectin-3
was predominantly found in the cytoplasm of quiescent fibroblasts while it was
intensively localized to the nucleus o f proliferating cultures of the same cells (120).
Galectin-3 can be phosphorylated by casein kinase I (121). Non-phosphorylated
galectin-3 was exclusively found in the nucleus, while phosphorylated galectin-3
was found both in the nucleus and cytoplasm (122). It is suggested that
phosphorylation may be important for nuclear export o f galectin-3. Cell surface
galectin-3 has been implicated in cell adhesion, whereas intracellular galectin-3
binds to RNA in nucleus through its N-terminal RNA binding domain (123).
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25
Activated macrophages, eosinophils, mast cells, epithelial cells of gastro-intestinal
and respiratory tracts as well as many different types o f tumor can over-express
galectin-3 (124,125,126,127,128, 129,130).
In the adult murine system, galectin-1 is abundant in tissues that originate
from mesoderm while galectin-3 is rich in epithelial cells, cartilage and lymphocytes.
Galectin-3 can bind to both intracellular and extracellular counter-receptors while all
galectin-1 counter-receptors identified so far are either on the surface of cells or
within ECM. However, the overlap in ligand specificity implies that galectin-1 and
galectin-3 may compete for binding to the same counter-receptors. They bind to
ECM proteins such as laminin, fibronectin, tenescin and integrins (131,132,133).
Galectins modulate cell-cell and cell-matrix adhesion.
Galectin-mediated effects on cell adhesion can be either inhibitory or
stimulatory. When galectins binds to the galactoside of cell surface integrins and
their extracellular interacting molecules, they may generate steric hindrance that can
further weaken the adhesive interactions between cells and matrix. However,
galectins can enforce the cell-to-cell and cell-to-ECM adhesion by simultaneous
binding to cell surface and matrix (134, 135,136). Galectin-1 inhibits the adhesion
of myoblast and other type o f cells to a laminin matrix, but other studies claim that it
can promote the adhesion of various cells to laminin and other ECM components
(137,138).
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26
Galectin-1 within ECM can mediate the adhesion o f ovarian carcinoma cells,
but increased levels o f galectin-1 on the surface of tumor cells inhibits adhesion to
ECM. The expression of specific counter-receptors may determine whether galectins
can mediate cell adhesion. The presence o f tumor specific antigen L3 can inhibit
galectin-3 mediated hemagglutination (131). CD98 is a membrane protein that
interacts with the cytoplasmic tails o f integrin subunits and can directly recruit
signaling molecules. Galectin-3 can binds to CD98 promoting its dimerization,
which in turn activates integrin through incompletely known signalling pathways
(139).
Galectins modulate cell proliferation and apoptosis.
I1 ......... ■■ — M ■ ■ ■ I H I ■■ III I M l III, m i HIIIMH M U ■ I I I ! ■ ■ ■ 'ft* '' — ■ « ■ ■ ■ ■■■■■■.^ "............
Galectins have also been found both to promote and inhibit cell proliferation.
The expression o f galectin-1 is associated with increased proliferation of rat
endothelial cells and transformation o f 3T3 fibroblasts, but exogenous galectin-1 can
inhibit the proliferation of mouse embryonic fibroblasts (140). Galectin-3 was
induced when T cells were activated by mitogenic CD3 antibody, IL-2, IL-4 and IL-
7, and the inhibition of galectin-3 expression made cells less responsive to these
mitogenic stimuli (141,142). Human breast cancer cells transfected with antisense
galectin-3 cDNA showed significant decrease in cell proliferation (143).
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27
In human lung fibroblasts, recombinant galectin-3 could stimulate DNA synthesis as
well as cell proliferation in a dose-dependent manner. In addition, this mitogenic
activity o f galectin-3 could be suppressed by adding lactose to the culture (144).
Galectin-1 can induce apoptosis of human thymocytes. The susceptible
populations for this apoptosis are CD3-CD4-CD8 double negative and double
positive thymocytes, indicating the involvement of galectin-1 in TCR mediated
negative selection (145). Galectin-1 also can induce apoptosis o f mature peripheral T
cells by a poorly understood mechanism distinct from Fas/FasL pathway (146).
In contrast to galectin-1, galectin-3 prevents apoptosis (147,148). When
galectin-3 DNA was transfected into leukemic T cells, they showed resistance to
apoptosis induced by Fas ligation and staurosporine (149). Over-expression of
galectin-3 in human breast carcinoma cells inhibited cisplatin-induced apoptosis and
increased survival rate against various apoptotic stimuli, including cyclo-
heximide/TNF-a and UVB irradiation (150). Galectin-3 has also been shown to
protect cells against apoptosis induced by loss of cell anchorage (151). A recent
study suggests that the phosphorylation of galectin-3 is required for its anti-apoptotic
function (152). In galectin-3 knockout mice, peritoneal macrophages treated with
INF-y and LPS died more rapidly than ones from wild-type mice (153). Galectin-3
has significant sequence homology to Bcl-2 (154).
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28
Both proteins are rich in Pro, Gly, and Ala in N-terminus and possess an
NWGR quartet in the C-terminus (150). Like other Bcl-2 family members, which
form heterodimers with Bcl-2, galectin-3 binds Bcl-2 in vitro (149). Since increased
cell adhesion is known to protect cells from apoptosis, galectin-3 mediated cell
adhesion might be responsible for the resistance to apoptosis. Also, it is possible that
galectin-3 protects cells from apoptosis through binding to cell surface
glycoconjugates. The mechanism underlying anti-apoptotic property of galectin-3
still remains to be elucidated.
Galectin-3 in inflammation
Since galectin-3 is known as an antigen expressed on the surface of activated
macrophages, its role in inflammation has been well investigated. In addition to its
function in cell adhesion and migration, it plays a role in regulating respiratory burst
and lymphocyte chemotaxis. Upon interaction with microorganisms and
inflammatory mediators, neutrophils produce huge amounts of cytotoxic superoxide
anion and hydrogen peroxide by activation o f the NADPH-oxidase. When peripheral
nuetrophils are primed with cytochalasin B, a substance that disrupts the
microfilament system, its NADPH-oxidase is activated by galectin-3 (155).
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29
Also, exudation-related activation increased neutrophil responsiveness to galectin-3.
Galectin-3 knockout mice significantly reduced inflammatory cell infiltration in the
peritoneal cavity in response to thioglycollate broth treatment because o f low number
of macrophages due to increased level of apoptosis and low NF-kB response of
inflammatory cells (153).
Galectin-3 in tumorigenesis and metastasis
Evidence suggests that expression o f galectin-3 is related to tumor formation
and metastasis. In the last decade, galectin-3 has been found in many epithelial
tumors such as melanoma, colon, thyroid, gastrointestinal, lung, breast and ovarian
carcinoma (156,157,158,159). Although the effects of galectin-3 on cell
proliferation, survival, and inflammation could influence growth and tumorgenesis,
the possible effect of galectin-3 in metastasis has gained attention due to its function
in regulating cell adhesion, cell motility, angiogenesis and invasive potential (160,
161,162). The metastatic potential of mouse melanoma and sarcoma was positively
correlated with the expression of galectin-3 on the cell surface (163). Transfection of
galectin-3 antisense oligonucleotides into metastatic colon cancer cells reduced liver
colonization and spontaneous metastasis in athymic mice (164).
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30
Similarly, oral administration o f modified citrus pectin, an inhibitor of galectin-3,
inhibits spontaneous metastasis in a rat prostate cancer model and anti galectin-3
antibody reduced liver metastasis o f adenocarcinoma in nude mice (165, 166).
Recent studies suggest that the adhesion of tumor cells on vascular endothelium does
not exactly follow the way of lymphocytes utilizing members o f selectin family to
recognize carbohydrate ligands (167, 168). Studies with human breast and prostate
cancer cells have shown that galectin-3 initiated the adhesion between tumor cells
and endothelium by specific interaction with Thomsen-Friedenreich (TF) antigen
(169,170).
TF antigen is a tumor antigen of epithelial cancers. In normal tissues, it is
hidden in other longer carbohydrate chains, but its hidden core-1 structure is exposed
during tumorgenesis (171). Highly metastatic breast carcinoma cells upregulated
galectin-3 and TF antigen, and increased adhesion to monolayer o f endothelial cells.
Soon after their adhesion, they enhanced homotypic adhesion to form multicellular
aggregates. Adding synthetic TF antigen antagonist inhibited this adhesion. In
addition, galectin-3 stimulates capillary tube formation of human umbilical vein
endothelial cells in vitro and angiogenesis in vivo (172). Galectin-3 plays a key role
in tumorigenesis and metastasis by increasing cell proliferation, inflammatory
reactions and anti-apoptosis and/or inducing homotypic and heterotypic aggregation
of cells and matrix (173,174).
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HYPOTHESIS
31
Ovarian cancer is a leading cause o f gynaecologic cancer mortality in US.
Hereditary change in genes such as BRCA1 or BRCA2, pregnancy, age and
hormones affect the incidence of this cancer. Ovarian cancers can arise from any
type of cells making up the ovary and they are classified as epithelial, stromal and
germ cell tumors. Approximately 90% o f ovarian cancers are epithelial in origin.
They are sub-classified into invasive and metastatic carcinoma, benign cystadenoma
and low malignant potential (LMP) tumors (175). Ovarian cancers usually spread by
direct extension to nearby organs or by seeding and shedding into the peritoneal
cavity even though they can invade lymphatic vessels or blood circulatory system.
Long-term cultures o f cystadenoma and LMP ovarian epithelial tumors were
previously established in our laboratory by introducing SV40 large T antigen (LTA)
into primary explanted cultures derived from such tumors (176). One such cell line
derived from papillary serous cystadenoma is called ML-10, and it was kept in
culture through its crisis period, which began after about 50 population doublings.
Spontaneous recovery from this crisis generated two clones, called MCY 39 and
MCV50 (177). This provides us with a longitudinal model for ovarian cancer
progression in vitro.
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32
My first goal when I began my thesis work was to examine how
ubiquitylation may be associated with ovarian epithelial tumor progression.
Since ubiquitylation affects the level o f intracellular proteins as well as their
localization and function, I reasoned that comparing ubiquitylated proteins, either
hyper- or hypo-ubiquitylated proteins, in mortal ML-10 cells to those in MCV-39
and MCV-50 cells could help us identify changes in protein ubiquitylation associated
with ovarian tumor progression. This led to the observation that nucleolin is
monoubiquitylated in these cells. My hypothesis is that the monoubiquitylation
modulates the function and localization o f nucleolin and it is associated with other
known post-translational modifications o f nucleolin such as phosphorylation.
Much of my thesis work focused on confirming this information and on examining
its functional significance. This work is presented in chapter 13.1 also studied the
difference in sumoylation between ML-10 cells and MCV-50 cells that is described
in chapter D E I. Another component o f my work has focused on the role of galectin-3
in mediating metastatic spread of ovarian carcinomas. This work is too premature to
be presented in this thesis, but the background information was reviewed in the
introduction.
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CHAPTER II
33
POST-TRANSLATIONAL MODIFICATION OF NUCLEOLIN BY
MONOUBIQUITYLATION IN HUMAN OVARIAN CARCINOMA CELLS
ABSTRACT
Ubiquitin is a small eukaryotic protein that is conjugated to lysine residues on
target proteins. Proteins with polyubiquitin chains, in which lysine 48 of ubiquitin
binds to the C-terminal glycine of the next ubiquitin molecule, are degraded by the
26S proteasome complex. Monoubiquitylation, single ubiquitin conjugation without
polymer chain formation, does not result in protein degradation.
Recent studies on histones, plasma membrane proteins in endocytosis, and
Gag proteins involved in retrovirus budding suggest that monoubiquitiylation may
regulate the location and function of its conjugating proteins. Here we report that
Nucleolin, a major eukaryotic phosphoprotein responsible for ribosome biogenesis
and maturation, is monoubiquitylated in various human ovarian carcinoma cell lines
and that the majority of monoubiquitylated nucleolins exist in the nuclear
compartment.
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34
Since nucleolin is regulated by various kinases, we sought to determine
whether the monoubiquitylation of nucleolin is related to its state of phosphorylation.
Phosphorylated and non-phosphorylated proteins from ovarian carcinoma cells were
separated by column chromatography and immunoprecipitated with an antibody
against ubiquitin. The only form o f monoubiquitylated nucleolin found in the
cytoplasm was phosphorylated while phosphorylated and non-phosphorylated forms
were both present in the nucleus, with a predominance o f the non-phosphorylated
form. In addition, a monoubiquitylated and phosphorylated 80kDa fragment of
nucleolin thought to be involved in initiation o f apoptosis was observed in the
cytoplasm.
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INTRODUCTION
35
In eukaroytic organisms, proteins are targeted for regulated degradation by
covalent attachment of the small, highly conserved protein called ubiquitin. In a
multistep reaction involving intricate enzymatic machinery, several molecules of
ubiquitin are attached to the target protein in a multimeric chain, which then serves
as a recognition signal for a large intracellular protease, the 26S proteasome.
Ubiquitylation is also emerging as a signal for various proteasome-independent
functions. For example, modification o f a plasma membrane protein by a single
ubiquitin moiety can trigger endocytosis and uptake into vacuoles. In addition,
monoubiquitylation ofhistones affects chromatin structure and contributes to the
regulation of gene expression. Monoubiquitylation is also important for retrovirus
budding as well as for maintaining DNA integrity in normal cells (40).
Nucleoli of rapidly dividing cells such as cancer cells are enlarged and
hyperactive in ribosome biogenesis. Nucleolin is a major eukaryotic nucleolar
phosphoprotein directly associated with ribosome biogenesis and cell proliferation
(178, 179, 180, 181). It increases ribosomal RNA (rRNA) synthesis and maturation.
It also induces the assembly of rRNA and ribosomal proteins to form ribosome
complexes (179,182). Nucleolin is shown to be predominantly intact among
nucleolar proteins in actively dividing cells, while it is fragmented by
autodegradation in non-dividing cells (183,184).
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36
The human nucleolin gene consists of 14 exons and 13 introns on
chromosome 2ql2 and its protein product contains 707 amino acids. Nucleolin is
often described as a 105 kDa protein because its acidic N-terminal Asp/Glu rich
domains reduce its electrophoretic mobility in SDS gels, giving the impression of a
larger molecular weight than expected for a protein of 707 amino acids (185, 186).
These highly acidic regions are separated from each other by basic sequences and
are analogous to high mobility group proteins, which can induce nucleolar chromatin
decondensation through ionic interactions with histone HI (187).
The acidic regions are responsible for the Ag-NOR stain of nucleolin (188).
The N-terminus of nucleolin binds to many different proteins such as ribosomal
proteins and U3 snoRNP (182). It can be phosphorylated by casein kinase (CK2)
(189), cyclin dependent kinase (CDK1) (190), protein kinase C (PKC-Q (191).
Phosphorylation of nucleolin by CK2 and CDK1 is tightly regulated during the cell
cycle, resulting in modulating the function and localization of nucleolin.
When CK2 phophorylates serine residues of nucleolin during interphase,
phosphorylated nucleolin, along with topoisomerase I (Topo I) and RNA polymerase
I (RNA pol I), localizes on chromosomes containing rDNA (192). CK2 mediated
phosphorylation of nucleolin can induce its proteolytic cleavage, resulting in 30 and
70kDa fragments that trigger rRNA transcription by RNA pol I (193).
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37
In regenerating rat hepatocytes and human tumor cells, the level of CK2 activity
phosphorylated nucleolin and rRNA synthesis are increased (194). Phosphorylation
of nucleolin by CK2 seems to be associated with cell growth, as hormones and
mitogens such as dexamethasone, androgen, epidermal growth factor and fibroblast
growth factor have been shown to induce the phosphorylation of nucleolin as well as
rRNA synthesis (195, 196,197,198). Insulin was shown to regulate phosphorylation
of nucleolin and RNA efflux from nuclei. Sub-nanomolar concentrations of this
hormone induce CK2-mediated phosphorylation of nucleolin and RNA efflux in
adipocytes, but micromolar concentrations lead to nucleolin dephosphorylation and
inhibition o f RNA efflux from nuclei (199).
During mitosis, threonines o f TPKK motifs that appear nine times in the N-
terminal domain of nucleolin are phosphorylated by CDK1 (200).
This phosphorylation is also closely associated with the degree of cell proliferation
and intracellular localization of nucleolin (201). Nuclear localization of nucleolin is
exclusively enhanced by dephosphorylation while its cytoplasmic translocation is
promoted by CDK1-mediated phosphorylation (202). Other kinases that
phosphorylate nucleolin include cyclic AMP-dependent protein kinase, protein
kinase C, and ecto-protein kinase (203). Although these kinases may regulate
biological functions of nucleolin in chromatin organization and ribosome assembly,
their exact roles are still unclear.
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38
The central globular domain of nucleolin has four RNA binding domains
(RBDs) also known as RNA recognition motifs (RRMs) (204). Nucleolin interacts
with RNA stem-loop structure and with RNA having 7-10 nucleotide loop (U/G
CCCGA) through its first two RBDs. Data on their binding affinity, specificity and
biological significance are limited, even if interactions between nucleolin and
multiple RNA targets have been suggested. The C-terminal domain possesses Arg-
Gly-Gly (RGG) repeats highly methylated on Arg residues (185,205). This domain
unfolds RNA secondary structure, which allows RNAs access to the central RNA
binding motifs o f nucleolin (206,207). In addition, it has the intrinsic protease
activity of nucleolin necessary for autodegradation (184).
Although nucleolin is a major nonhistone nucleolar protein, it is a ubiquitous
protein and its function is not limited to that o f ribosome biogenesis (208,209,210).
It acts as a shuttling protein between the plasma membrane, cytoplasm, and nucleus
(211). Through its nucleo-cytoplasmic shuttling property, nucleolin plays a role as
a carrier either during the import of ribosomal proteins to the nucleus or during the
export of ribosomal subunits to the cytoplasm (212,213). The bipartite nuclear
localization signal, located between N-terminal domain and central RNA binding
domain of nucleolin, is required for the import function of nucleolin (214). Nucleolin
and HSP70 contain nucleolar-targeting sequences homologous to the HIV tat protein.
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39
Nucleolin has been suggested as potential receptor for HIV due to its interaction with
the V3loop o f gpl20 (215). In fact, human nucleolin binds to hepatitis delta virus
and Coxsackie B viruses on the cell surface (216).
Since the synthesis o f nucleolin is positively related to the rate of cell
division, it is not surprising that many cancer cells show high expression level of
nucleolin. Nucleolin remains low in nondividing cells and is preferentially associated
with chromatin. The amount o f nucleolin is low and mainly localized in the nucleus
in serum-deprived cells (217). Its expression is induced by v-src during mid and late
G l, which in turn activates transcription of ribosomal genes. Thus, the upregulation
o f nucleolin seems to be necessary for G0-G1 cell cycle progression (195). Nucleolin
is down regulated along with N-myc and HSP70 during differentiation of human
neuroblastoma (218).
Many nuclear proteins such as B23, topoisomerase I (Topo I), midkine
and heparin binding growth-associated molecules bind to nucleolin. Interaction of
nucleolin with B23 is regarded as a nucleolar targeting mechanism for nucleolin
(219). Topo I regulates DNA supercoiling, gene transcription, and rDNA
recombination. The interaction o f Topo I with nucleolin targets Topo I to enter the
nucleus (220). Nucleolin also plays a central role in lymphocyte-mediated apoptosis.
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40
When granzyme A released from cytotoxic T cells or natural killer cells cleaves
nucleolin in target cells, the 80 and 37 kDa fragments of nucleolin can activate
endonucleases in order to initiate DNA fragmentation (221).
A previous student in our laboratory developed an in vitro tissue culture
model for ovarian cystadenomas by introducing an adenoviral vector containing
SV40 large T antigen into primary explanted cultures of these benign tumors.
One of the resulting cell strains, called ML-10, gave rise to two spontaneously
immortalized cell clones called MCV-39 and MCV-50 (177).
I used this in vitro longitudinal model o f ovarian tumor progression to
investigate how ubiquitylation may affect the progression of ovarian cancer.
Here, I report that nucleolin is mono-ubiquitylated in our in vitro model system as
well as in various ovarian carcinoma cell lines. This post-translational modification
appears to be associated with phosphorylation and intracellular localization of
nucleolin.
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MATERIALS AND METHODS
41
Cell lines
The ML-10 cell strain was established from a primary culture of a human
papillary cystadenoma by transfection with an adenovirus vector expressing SV40
large T antigen (176). MCV-39 and MCV-50 are immortal cell lines spontaneously
derived from ML-10 (177). MCV-152 cells were established by transfection of ML-
10 with an expression vector for the telomerase catalytic subunit. The above cells
were grown in MEM (Invitrogen Corporation, Carlsbad, CA) supplemented with
10% fetal bovine serum (FBS). CAOV3, HEY, HOC7 ovarian carcinoma cells were
obtained from Dr. Ronald Buick, University o f Toronto (222), and SKOV3 (ATCC #
HTB77) human ovarian carcinoma cell line were purchased from the American Type
Culture Collection. IOSE 80 is a human ovarian surface epithelial cell immortalized
by expressing SV40 large T antigen (obtained from Dr. Nellie Aversperg, Univ. of
British Columbia). These cells were grown in DMEM (Invitrogen Corporation,
Carlsbad, CA) supplemented with 10% FBS.
Protein extraction
Ceils were grown in 15 cm tissue culture dishes until confluence. In the cold
room, cell layers were collected by scraping and transferred to a 50 ml centrifuge
tube. Cell pellets were obtained by centrifugation at 4000 rpm for 5 minutes.
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42
The pellets were washed twice with ice cold PBS and lysed with 1.25 ml Triton-lysis
buffer (25mM Na-phosphate, 150mM NaCl, 1% Triton X-100, 5mM EDTA pH 8.0,
50mM NaF, aprotinin and leupeptin) by incubation on ice for 20 minutes. The
extracts were transferred to a 1.5 ml ependorf tube and centrifuged at 12000 rpm for
20 minutes in a cold room. Supernatants were stored in a -8 0 0 C for further assay.
To obtain nuclear extracts, cell layers were collected by scraping and
transferred to a 50 ml centrifuge tube in the cold room. Cell pellets were obtained by
centrifugation at 4000 rpm for 5 minutes and were washed twice with ice cold PBS
and lysed with 2.5 ml buffer A (lOmM HEPES, 1.5mM MgCk, lOmM KC1,0.5mM
DTT, 0.1 % Triton X-100, 0.5mM PMSF, lOmM P-glycerophosphate) by 20 minute
incubation on ice. Cell extracts were centrifuged at 5000 rpm for 10 minutes. The
supernatant was collected as cytoplasmic protein extracts, and nuclear pellets were
carefully resuspended in 0.5 ml buffer C (20mM HEPES, 25% glycerol, 0.42M
NaCl, 1.4mM MgCk, 0.2mM EDTA, 0.5mM PMSF, 1 pg/ml leupeptin and
aprotinin, lOmM P-glycerophosphate). After incubation o f nuclear lysates at 4 0 C in
a rocker for 30 minutes, nuclear proteins were obtained by centrifugation at 4 0 C for
30 minutes at 13000 rpm. Both cytoplasmic and nuclear protein extracts were stored
in a -8 0 °C .
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43
Immunoprecipitation and Immunoblotting analyses
Antibodies used were anti-Ubiquitin (FL-76, Santa Cruz) polyclonal, anti-
Nucelolin (MS-3, Santa Cruz) monoclonal, anti-HSP 60 (H4149, Sigma) mono
clonal, anti-Phospho-p44/42 Map Kinase (9101, Cell Signalling) polyclonal, anti-
Histone H3 (9712, Cell Signalling) polyclonal antibodies. Immunoprecipitation was
performed with 400 pg cellular proteins and 2 pg primary antibodies, which were
incubated in 1 ml lysis buffer at 4 °C for 2 hours. Then 50 pi of resuspended volume
o f protein AorG agarose conjugate were added to the above reaction and incubated
at 4 0 C overnight. Immunoprecipitates were collected by centrifugation at 2500 rpm
for 5 minutes at 4 0 C. The supernatants were discarded and pellets were washed 4
times with 1 ml lysis buffer. After the final wash, the supernatant was removed and
the pellet was resuspend in 20 pi o f 2X electrophoresis sample buffer. The samples
were boiled for 5 minutes and analyzed by SDS-PAGE. Immunoblotting was
performed with overnight electrotransfer to PVDF membrane (Bio-Rad) and 5 %
non-fat dry milk in TBST blocking buffer. ECL reagent and ECL-Hyperfilm
(Amersham Pharmacia Biotech, Piscataway, NJ) were used for detection.
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44
Two-dimensional gel electrophoresis
Isoelectric focusing was carried out in glass tubes of inner diameter 2 mm
using 2% pH 3.5-10 (Amersham Pharmacia Biotech, Piscataway, NJ) for 9600 volt-
hrs. 1 pg of IEF internal standard, tropomyosin, was added to each sample. This
protein migrates as a doublet with lower polypeptide spot o f MW 33,000 and pi 5.2.
After equilibration for 10 minutes in buffer ‘O’ (10% glycerol, 50mM dithiothreitol,
2.3% SDS and 0.0625 M Tris, pH 6.8) the tubes were sealed at the top o f stacking
gels on top of 10% acrylamide slab gels. SDS slab gel electrophoresis was carried
out for about 4 hours at 12.5 mA/gel. After slab gel electrophoresis, the gel was
transblotted onto a PVDF membrane in transfer buffer (12.5mM Tris, pH 8.8, 86mM
glycine, 10% methanol) overnight at 200mA and approximately 100 volts/two gels.
Phosphoprotein Purification
Phosphoprotein purification was performed as described in the Phospho-
Protein Purification Kit handbook (37101, QIAGEN). 1.5 x 107 of HEY cells were
lysed in 5ml o f phospho-protein lysis buffer containing 0.25 % CHAPS, a protease
inhibitor, and benzonase and incubated for 30 minutes at 4 °C. It was vortexed
briefly every 10 minutes. After incubation, the cell lysate was centrifuged at 10,000 x
g and 4 °C for 30 minutes. 2.5 mg o f protein were taken and adjusted to 0.1 mg/ml
by adding phospho-protein lysis buffer containing 0.25 % CHAPS.
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45
The lysate was transferred into the phosphor-purification column. The flow-through
fractions were collected for analysis o f unphosphorylated proteins. 500 fil
phosphoprotein elution buffer containing 0.25 % CHAPS was applied to collect the
eluted fraction. Both the flow-through fraction and the eluted fraction were
concentrated by TCA protein precipitation protocol.
GST-Nucleolin gene fusion system
Full-length nucleolin cDNA (obtained from Dr. B. C. Valdez, Baylor College
of Medicine, Houston, TX) was inserted into pGEX-6P-l expression vector (1056-
1034, Amersham Pharmacia Biotech). BL21-CodonPlus (DE3)-RIL competent cells
(230245, Stratagene) were transformed with pGEX-6P-1 /nucleolin plasmid and
plated on LBAG plates. After confirmation o f the proper orientation and junction,
fusion protein expression was optimized by modulating the incubation time and
temperature (37 °C), and the concentration oflPTG. The bacterial cell pellets were
lysed by sonication and further purified by MicroSpin GST purification module
(27-4570-03, Amersham Pharmacia Biotech). The purified GST-Nucleolin was
analysed by immunoblotting with antibodies against GST (27-4577-01, Amersham
Pharmacia Biotech) and Nucleolin (MS-3, Santa Cruz) after electrophoresis on SDS-
PAGE. Nucleolin was released from GST-Nucleolin by adding two units of
PreScission protease (27-0843-01, Amersham Pharmacia Biotech).
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RESULTS
46
Differential expression of ubiquitylated nucleolin in ovarian tumor cells at
various stage of progression.
Total protein extracts from MCV-152, MCV-50, MCV-39, and ML-10 were
first analyzed by immunoblotting using an anti-ubiquitin antibody to look for
differences in protein ubiquitylation in those various cells, which were all derived
from the same benign ovarian tumor and can be regarded as representing different
stages o f tumor progression. These initial experiments revealed no detectable
differences between the various cell lines (data not shown). We then focused on
nuclear extracts from each cell line. Immunoblots of the nuclear proteins revealed
two protein fragments with respective sizes of approximately 35 kDa and 105kDa,
which were significantly upregulated and ubiquitylated in the three immortalized cell
lines compared to the mortal ML-10 cells (Fig. 1).
Nuclear proteins from ML-10 and MCV-50 were examined by two-
dimensional gel electrophoresis followed by immunoblotting with antibody against
ubiquitin to further examine differences in protein ubiquitylation between these two
cell lines. Although low molecular weight proteins were well resolved, high
molecular weight proteins above 100 kDa showed considerable overlap in the two
dimensional gel. Mass-spectrometry analysis suggested that the 35kDa protein might
be either an unknown protein or OZF (Only Zinc Finger) transcription factor, but
exposure o f the same membrane to antibody against OZF ruled out the possibility.
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M CV MCV MCV ML
152 50 39 10
47
105kDa
35kDa — W - ^
Histone H3
Figure 1. Differential ubiquitylation of nuclear proteins in an in vitro longitudinal
model for ovarian cancer progression. Nuclear proteins of ML-10, MCV-39, MCV-
50 and MCV-152 were separated by SDS-PAGE and imnaenoblotted with antibody
against ubiquitin, 105 kDa protein in MCV-39 and MCV-50 was more ubiquitylated
than in ML-10 and MCV-152, while 35 kDa protein in MCV-39, MCV-50 and
MCV-152 was more ubiquitylated than in ML-10. As loading control, the membrane
was reprobed with anti-histone H3 antibody.
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48
To identify 105 kDa protein, nuclear extracts o f ML-10 and MCV-50 were
immunoprecipitated by ubiquitin antibody and electrophoresed on SDS-FAGE.
The 105 kDa protein was visualized by coomassie blue stain. The band was isolated
by cutting out gel and was used for amino acid sequence analysis. Identity of 105
kDa protein was discovered to be human nucleolin and the immunoprecipitation with
antibody against ubiquitin followed by immunoblotting with antibody against
nucleolin confirmed this (Fig. 2).
Since nucleolin behaves as a 105 kDa protein in SDS-PAGE and one
ubiquitin molecule has a mass o f 8.5kDa, the presence of a single ubiquitylated
nucleolin band with an apparent molecular weight of about 105 kDa suggests that the
ubiquitylated nucleolin fragment in the above experiment was monoubiquitylated.
This protein modification is not restricted to the components of our in vitro model or
to cells expressing SV40 large T antigen because immunoprecipitation o f nuclear
extracts from five established epithelial ovarian carcinoma cell lines with anti-
ubiquitin antibody followed by immunoblotting with anti-nucleolin revealed the
presence of this protein in the immunoprecipitates from all the cell lines (Fig. 3 A).
While the amounts o f nuclear ubiquitylated nucleolin varied among the different cell
lines, the total amounts of nucleolin were almost identical (Fig. 3B).
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49
Figure 2. Identification of the 105 kDa ubiquitylated fragment as nucleolin. Nuclear
extracts of ML-10, MCV-39, MCV-50, and MCV-152 were immunoprecipitated
with anti-ubiquitin antibody and electrophoresed on SDS-PAGE. Separated proteins
were transferred to PVDF membrane and were immunoblotted with anti-ubiquitin
antibody (A). Immunoprecipitation with anti-ubiquitin antibody followed by
immunoblotting with anti-nucleolin antibody (B) confirmed the result from protein
sequencing analysis indicating that the 105 kDa protein is nucleolin. NC is negative
control that doesn’t have any protein extract.
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ML
10
MCV MCV MCV
39 50 152
105 k D a ►
B
MCV-50 M L -10
105 kDa
N.C.
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5!
Figure 3. Differential expression o f monoubiquitylated nucleolin in various human
ovarian cell lines. Nuclear proteins o f ML-10, MCV-50, CAOV3, HEY, HOC-7,
IOSE 80, and SKOV3 were immunoprecipitated with anti-ubiquitin antibody and
immunoblotted with anti-nucleolin antibody. NC stands for negative control that
lacks any cellular proteins (A). Whole cell extracts of ML-10, MCV-50, CAOV3,
HEY, HOC-7, IOSE 80, and SKOV3 were immunoblotted with anti-nucleolin
antibody without prior immunoprecipitation to evaluate the total amounts of
nucleolin in the various cell lines (B).
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A
52
ML-10 MCV-50 CA0V3 HEY HOC-7 IOSE80 SKGV3 NC
105 kDa -►
B
ML-10 MCV-50 CAOV3 HEY HOC-7 IOSE80 SKOV3
105 kDa
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53
Monoubiquitylation is associated with phosphorylation and the localization of
nucleolin in ovarian cells.
As a major ubiquitous nucleolar phosphoprotein, the function and
localization o f nucleolin is regulated by phosphorylation (201). Upon mitogenic
stimulation, the serines o f nucleolin, topoisomerase I, and RNA polymerase I are
phosphorylated by casein kinase (CK) 2. Those phosphorylated proteins localize on
chromosomes to initiate rDNA transcription in the G1 phase (192).
During mitosis, the threonine o f nucleolin is phosphorylated by CDK1 (200).
It appears - although largely hypothetical - that consecutive CDK1 and CK2
phosphorylations on nucleolin may regulate nucleolar structure and activities in
dividing cells.
We hypothesized that there may be cross talk between phosphorylation and
monoubiquitylation o f nucleolin. To test this hypothesis, protein extracts from HEY
ovarian carcinoma cells were subjected to affinity chromatography to separate
phosphorylated from unphosphorylated proteins. Complete separation of
phosphorylated and unphosphorylated proteins was confirmed by immunodetection
of phosphorylated p44/p42 MAPK (Fig. 4A). These two protein fractions were
immunoprecipitated with anti-ubiquitin antibody followed by immunoblotting with
anti-nucleolin antibody. The result showed that monoubiquitylated nucleolin was
found primarily in the unphosphorylated protein population (Fig. 4B).
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54
Given that phosphorylation can promote cytoplasmic localization of
nucleolin, we sought to investigate whether monoubiquitylation and phosphorylation
interact to influence the localization of nucleolin. Cytoplasmic and nuclear proteins
extracted from HEY cells were further separated into phosohorylated and
unphosphorylated proteins, resulting in four different fractions. Each fraction was
immunoprecipitated with an antibody against ubiquitin followed by immunoblotting
with an antibody against nucleolin (Fig. 4C). The only ubiquitylated nucleolin found
in the cytoplasm was phosphorylated while most of the ubiquitylated nucleolin found
in the nucleus was unphosphorylated (Fig. 4C). Examination of the state of
phosphorylation of total nucleolin in the fractions showed that most of the
cytoplasmic nucleolin was phophorylated while nuclear nucleolin was divided about
equally into phosphorylated and unphosphorylated forms (Fig. 4D).
In addition, the 80kDa nucleolin fragment was predominantly located in the
cytoplasmic fraction, and it was the only fragment modified by both
monoubiquitylation and phosphorylation although there were other smaller nucleolin
fragments (Fig. 4D).
The results also showed that the 80 kDa nucleolin fragment, a product of
autodegradation, was only present in the cytoplasm. Although most of this fragment
was phosphorylated, an unphosphorylated form was also detected (Fig. 4D).
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55
Nucleolin that was monoubiquitylated and phosphorylated was the major
modified form in the cytoplasm, while forms modified by phosphorylation,
ubiquitylation or both were found in the nuclear fraction.
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56
Figure 4. Association between nucleolin monoubiquitylation and phosphorylation in
ovarian cells. Nuclear and cytoplasmic protein extracts from HEY cells were further
divided into phosphorylated and unphosphorylated fractions. Immunoblotting with
antibody against phosphorylated p44/42 MAPK was used to verify the efficiency of
phosphoprotein fractionation from whole cell extracts (A). Whole cell extracts of
HEY were immunoprecipitated with anti-ubiquitin antibody followed by
immunoblotting with anti-nucleolin antibody, showing that the monoubiquitylated
nucleolin was predominantly unphophorylated (B). Cytoplasmic and nuclear proteins
o f HEY were further separated into phosohorylated and unphosphorylated proteins,
resulting in four different fractions o f proteins: cytoplasmic unphosphorylated (Cyt
UP), cytoplasmic phosphorylated (Cyt P), nuclear phosphorylated (Nuc P) and
nuclear unphosphorylated (Nuc UP). The fractions were immunoprecipitated with an
antibody against ubiquitin followed by immunoblotting with an antibody against
nucleolin(C). Cytoplasmic unphosphorylated (Cyt UP), cytoplasmic phosphorylated
(Cyt P), nuclear phosphorylated (Nuc P) and nuclear unphosphorylated (Nuc UP)
protein fractions were immunoblotted with an antibody against nucleolin without
prior immunoprecipitation to examine the state o f phosphorylation of total nucleolin
in each fraction. Arrows indicate 105kDa intact nucleolin and 80kDa nucleolin
fragment (D).
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A
57
105 kDa .
80 kDa.
Phos.
p44/42 MAPK .
B
Phos.
Nucleolin
Cyt UP Cyt P Nuc P
D
Cyt UP Cyt P Nuc P
105 kDa_____*. • ;
80 kDa * » .
UnPhos.
UnPhos.
Nuc UP
illiiiiil'
Nuc UP
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58
Preparation to identify nucleolin monoubiquitylation site
Lys residues make up approximately 12 % of the total amino acids of
nucleolin and broadly extend from N-terminal to C-terminal ends. Bacterial GST-
Nucleolin fusion protein lacking any post-translational modification (Fig. 5 A, B, C)
and monoubiquitylated nucleolin from MCV 50 cells were purified. To define a
specific Lys residue responsible for the monoubiquitylation, those proteins will be
analyzed by the mass-spectrometry (work in progress).
When nucleolin was immunoprecipitated with antibody against nucleolin, the
monoubiquitylated nucleolin was rarely detected while immunoprecipitation with
antibody against ubiquitin pulled down significant amounts of mono-ubiquitylated
nucleolin from the same protein extracts (Fig. 6). The above observation leads us to
assume that monoubiquitylation may induce a conformational change in nucleolin
that may screen the antigen binding sites o f the modified nucleolin.
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59
Figure 5. Expression and purification o f GST-Nucleolin fusion protein. PGEX6p-
1/Nucleolin plasmid was transfected into BL-21 competent cells and whole proteins
were extracted by sonication. Those proteins were electrophoresed on SDS-PAGE
and immunoblotted with anti-nucleolin antibody. Due to its large size, the major
fusion protein is partially degraded to 50kDa fragment (A). Full length GST-
Nucleolin was produced by transfecting pGEX6p-1 /Nucleolin plasmid to BL21 -
CodonPlus competent cells. 1 to 4 indicate pGEX6p-l /Nucleolin plasmid
transfected bacteria while 5 to 8 indicate pGEX6p-l plasmid vector transfected ones.
Two different temperatures (room temperature: 1,2, 5 and 6, and 37 degree: 3 ,4 , 7
and 8) and IPTG induction (with induction: 1, 3,5, and 7 and without induction: 2 ,4 ,
6, and 8) were used to optimize the conditions. Clone 3 showed a positive fusion
protein (arrow) that was authenticated by immunoblots with anti-GST and anti-
nucleolin antibodies (B). After purification from this clone, the GST fusion tag was
cleaved by a Prescission protease (C). Both enzyme-processed (cut) and -
unprocessed (uncut) GST-Nucleolins were assayed on SDS-PAGE and
immunoblotted with antibodies against nucleolin and GST. Processed GST-
Nucleolin shifted down to 105 kDa while unprocessed GST-Nucleolin remained at
140 kDa, which is equivalent to the sum of GST and nucleolin molecular weights.
Anti-GST antibody only reacted with the unprocessed GST-Nucleolin, most likely
because o f conformational changes induced by GST.
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A
60
1 2
cut
m
' ■ A
P k
i s t
6 7 8
Nuc Ab
GST Ab
Nuc Ab
III*
#6 t>k
f c - f c
GST Ab
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61
Figure 6. Monoubiquitylation may induce a conformational change in nucleolin.
Protein extracted from HEY cells was immunoprecipitated either with monoclonal
anti-nucleolin antibody (MS-3)(1 and 2) or polyclonal anti-nucleolin antibody
(FL-18)(3 and 4). Negative controls (NC) (1 and 3) used the same reaction
conditions but lacked any protein extracts. Nucleolin (arrow) was detected by
coomassie blue staining and by immunoblotting with monoclonal anti-nucleolin
antibody (MS-3) (A). Nucleolin immunoprecipitated by anti-nucleolin antibodies
was immunoblotted with anti-ubiquitin antibody (FL-76). Compared to the signal of
monoubiquitylated nucleolin detected after immunoprecipitation with anti-ubiquitin
antibody, that obtained after immunoprecipitation with anti-nucleolin antibodies was
much weaker (B). Arrow indicates detectable amount of mono-ubiquitylated
nucleolin obtained by immunoprecipitation with monoclonal anti-nucleolin antibody
(MS-3) that is much less than the monoubiquitylated nucleolin by immuno-
precopitation with anti-ubiquitin antibody (FL-76). Two negative controls, NCI
(same condition without protein extract) and NC2 (same condition without anti-
ubiquitin antibody), were used.
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62
MS-3 FL-18
NC HEY NC HEY
MS-3 FL-18
NC HEY NC HEY
105 kDa
< ► '
IP with anti-nucleolin Abs IP with anti-nucleolin Abs and
(Cootnassie Blue Stain) WB with anti-nucleolin Ab
B
MS-3 FL-18
NC HEY NC HEY NCI NC2 HEY
105 kDa •
105 kDa •
■ J 8
IP with anti-nucleolin Abs and IP with anti-ubiquitin Ab and
WB with anti-ubiquitin Ab WB with anti-nucleolin Ab
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DISCUSSION
63
This study clearly suggests that the nucleolin which runs on SDS-PAGE as a
105 kDa band is monoubiquitylated in various human ovarian carcinoma cell lines.
The results also suggest that this post- translational modification might be associated
with another important modification, phosphorylation, in order to regulate the
intracellular localization of nucleolin.
Our study began by comparing the ubiquitylated proteins in a panel of
ovarian tumor cell lines derived from the same human tumor and showing different
degrees of malignant transformation. The finding that nucleolin ubiquitylation was
more abundant in immortalized cells than in the mortal parental cells in this in vitro
model suggests a possible association between this post translational modification of
nucleolin and tumor progression.
Accurate distinction between mono- and poly-ubiquitylation was crucial in
this study. Although such distinction cannot be made with any direct assay, poly-
ubiquitylation typically increases the molecular weight o f target proteins on SDS-
PAGE, resulting in multiple bands above its original molecular weight.
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64
Because nucleolin behaves as 105 kDa protein and ubiquitylated nucleolin in ovarian
cells showed one single band without any substantial shift in apparent molecular
weight, we conclude that the ubiquitylated form o f nucleolin observed in these
studies was probably monoubiquitylated. Its presence in various established ovarian
carcinoma cell lines suggested that this modification is not limited to cells expressing
SV40 large T antigen such as those from our in vitro model of ovarian tumor
progression. Unlike polyubiquitylation, monoubiquitylation modulates the location
and function of target proteins, as discussed in chapter 1.
Ribosome biogenesis is one o f the most complex and meticulously regulated
cellular pathways. Nucleolin plays important roles in ribosome biogenesis by
influencing ribosomal RNA (rRNA) transcription, rRNA maturation and ribosome
assembly. Nucleolin has a negatively charged N-terminal domain, a nuclear
localization signal, four RNA binding domains, and a C-terminal GAR/RGG
domain. Its biological functions also include nucleo-cytoplasmic transportation,
transcription factor, DNA helicase, cell proliferation, angiogenesis, and apoptosis
(182,223). Nucleolin is highly phosphorylated and methylated, and also could be
ADP-ribosylated. The N-terminal domain o f nucleolin contains phosphorylation sites
for CK2, CDK1 and other protein kinases. CK2 mediated phophorylation and auto-
lytic cleavages o f nucleolin are necessary to initiate rRNA transcription.
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65
Intracellular localization o f nucleolin has been extensively studied. It is
generally accepted that the localization of this nuclear phosphoprotein is regulated by
phosphorylation. In Xenopus egg extracts, massive phosphorylation by CDK1 or
CK2 localized nucleolin to the cytoplasm. Translocation to the nucleus was seen
after dephosphorylation (202). I studied whether the monoubiquitylation is
associated with phosphorylation in order to modulate the localization of nucleolin.
When protein extracts from whole cell were divided into phosphorylated and
unphosphorylated proteins, monoubiquitylated nucleolin was most abundant in the
unphosphorylated fraction implying the majority o f monoubiquitylated nucleolin is
unphosphorylated. To determine whether the monoubiquitylation is associated with
the intracellular localization o f nucleolin, cell extracts were further fractionated into
cytoplasmic unphosphorylated protein, cytoplasmic phosphoprotein, nuclear
unphosphorylated protein and nuclear phosphoprotein. Nucleolin was either
modified by both monoubiquitylation and phosphorylation or was not modified by
either of those modifications in the cytoplasm. It existed in all possible combinations
in the nucleus. In addition to the 105 kDa form, nucleolin also exists in fragments of
various sizes in the cytoplasm. Our results showed that the 80 kDa nucleolin
fragment, which we found only in the cytoplasm, was either unmodified or was both
ubiquitylated and phosphorylated.
The above observations lead me to postulate the following model: In the
nucleus, nucleolin exists equally as monoubiquitylated or phosphorylated forms.
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66
When nucleolin is modified with both modifications, it acquires the ability to
translocalize to cytoplasm where it is further cleaved into an 80kDa fragment that
retains both modifications. Although nucleolin and its fragments without any of
those modifications may be in cytoplasm and nucleus, nucleolin with only one of
these modifications is not allowed to localize to cytoplasm (Fig. 7). Therefore,
monoubiqitylation and phosphorylation may synergistically induce cytoplasmic
translocalization o f nucleolin. The proteolysis o f nucleolin is a prerequisite for
lymphocyte-mediated apoptosis. Granzyme from cytotoxic T cells and natural killer
cells physiologically cleaves nucleolin o f target cells into small fragments, resulting
in activation of endonucleases to cause DNA fragmentation. It was demonstrated that
intact nucleolin is dominant in actively dividing cells while the cleaved forms of
nucleolin are dominant in non-dividing cells or cells in apoptosis (184,186).
Our results are at odds with these studies because we detected large amounts
o f intact nucleolin as well as cleaved nucleolin from actively proliferating MCV50,
HEY and IOSE 80. In addition, fragmented nucleolin is found in the
unphosphorylated fraction o f cytoplasmic proteins. The cytoplasm may be where the
degradation of nucleolin occurs because cleaved nucleolin is not seen In nucleus.
Our data also suggests that the phosphorylation may not be the only factor affecting
the stability of nucleolin. Monoubiquityltion might keep nucleolin from further
degradation.
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67
Although we still do not know if the monoubiquitylation of nucleolin is
reversible and what enzyme is responsible for this modification, monoubiquitylation
and phosphorylation may be closely associated with each other to modulate the
stability, function, and localization of nucleolin.
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68
j t s t p p X \ y-p-erz
^ + A
«£A> CAJ
Figure 7. Model for the role of monoubiquitylation and phosphorylation in
regulating intracellular localization of nucleolin. Nucleolin can be mono-
ubiquitylated, phosphorylated, or both. The nucleolin with both modifications is able
to translocalize to cytoplasm where it is cleaved into an SOkDa fragment with both
modifications. Although whole and fragmented nucleolin can be unmodified in the
cytoplasm and the nucleus, nucleolin with only one of these modifications is not
allowed in the cytoplasm. Ribbon represents nucleolin and sphere indicates
monoubiquitin. P is a phosphate.
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69
CHAPTER III
POST-TRANSLATIONAL MODIFICATION OF GRP78 BY SUMOYLATIQN
IN HUMAN OVARIAN CARCINOMA CELLS
INTRODUCTION
Glucose regulated proteins (GRPs) are upregulated when cells are starved of
glucose in their culture medium. The most commonly observed two GRPs in
mammals and chicken weigh 78 kDa and 94 kDa that are respectively called GRP78
and GRP94 (224). GRP78 is identical with immunoglobin heavy chain binding
protein (BiP) and shares a sequence homology with the heat shock protein 70
(HSP70) family. However, it is an acidic protein with pi of 5.2 and is distinguished
from HSP 70 on two-dimensional gel electrophoresis (225). GRP78 is normally
localized in the endoplasmic reticulum (ER), but various stress conditions such as
glucose depletion, cellular glycosylation blocking agents, viral infection, prolonged
anaerobiosis, low extracellular pH induce GRP78 expression as well as its
translocalization to the nucleus (226). As a molecular chaperone, GRP78 trasiently
binds to nascent polypeptide chains trasversing the ER membrane and facilitates
their correct folding, processing, assembly, glycosylation and degradation in ER
( 11).
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70
Because it protects cells against physiological stress-mediated apoptosis and
maintains cell homeostasis, abnormal regulation o f GRP78 has been found in
pathologic conditions (227). Its level is decreased in familial Alzheimer’s disease
and the downregulation of GRP78 was associated with missense mutations in the
human presenilin-1 gene (226). It has been suggested that induction of GRP78 is
related with the development of drug resistance to anticancer drugs (228).
GRP78 has ATPase activity that autophosphorylates a threonine residue
(Thr-229) (229). Another post-translational modification, ADP-ribosylation, of
GRP78 has been reported (230). Both modifications are diminished under conditions
where GRP78 is upregulated, suggesting that the active form is not modified (231).
In addition, the modified GRP78 appears to be dimeric while the ligand-bound
GRP78 is in a monomeric form (232).
Sumo protein has only about 18% sequence homology to ubiquitin and also
differs from it by possessing short N-terminal extensions. Sumoylation requires the
El heterodimer AOS1/UBA2 and E2 UBC9 (72, 75). Although several sumo
molecules can be covalently coupled to substrates, no poly-sumo chain can be
formed. Sumoylation does not cause degradation of its target proteins, but it has
close association with the ubiquitylation system. Sumo sometimes blocks
ubiquitylation and degradation of a substrate. Sumoylated IkBo c is resistant to
phosphorylation- ubiquitylation mediated proteasomal degradation, suggesting the
competition between sumoylation and ubiquitylation (83).
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71
A single Lys residue in the C-terminal region of p53, important for Mdm2 mediated
ubiquitylation of p53, is sumoylated and this sumoylation increases transactivation
ability of p53 (84). Sumo targets RanGAPl (Ras related GTPase activating protein)
to the nuclear pore and stabilizes the association of RanGAPl with RanBP2 (77).
Sumo restricts PML (promyelocytic leukaemia), a RING finger protein, to the
nuclear bodies during interphase, and lack o f this sumoylation leads to acute
promyelocytic leukaemia (79). Sumo also modifies the expression o f two
mammalian glucose transporters, GLUT land GLUT4. Overexpression of UBC9
downregulates GLUT1, but it upregulates GLUT4 (223). Sumo does not function
analogously to ubiquitin, and studies on sumo substrates suggest that it affects
cellular localization, stabilization, and protein-protein interaction o f target proteins.
Our laboratory developed an in vitro tissue culture model for ovarian
cystadenomas by introducing an adenoviral vector containing SY40 large T antigen
into primary explanted cultures of these benign tumors. One of the resulting cell
strains, called ML-10, gave rise to two spontaneously immortalized cell clones called
MCV-39 and MCV-50 (177). Investigation on this in vitro longitudinal model of
ovarian tumor progression led to the novel finding that nucleolin is mono-
ubiquitylated and this post translational modification is associated with
phosphorylation and intracellular localization of nucleolin. Here, we sought to see
the difference in sumoylation between ML-10 cells and the MCV-50 clone.
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72
MATERIALS AND METHODS
Cell lines
The ML-10 cell strain was established from a primary culture of a human
papillary cystadenoma by transfection with an adenovirus vector expressing SV40
large T antigen (176). MCV-50 is an immortal cell line spontaneously derived from
ML-10 (177). The above cells were grown in MEM (Invitrogen Corporation,
Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS).
Nuclear protein extraction
To get nuclear extracts, cell layers were collected by scraping and transferred
to a 50 ml centrifuge tube in the cold room. Cell pellets were obtained by
centrifugation at 4000 rpm, 5 minute and were washed twice with ice cold PBS and
lysed with 2.5 ml buffer A (lOmM HEPES, 1.5mM MgCfr, lOmM KC1, 0.5mM
DTT, 0.1 % Triton X-100, O .SmM PMSF, IQ roM p-glycerophosphate) by 20 minute
incubation on ice. Cell extracts were centrifuged at 5000 rpm for 10 minutes. The
supernatant was collected as cytoplasmic protein extracts, and nucleus pellets were
carefully resuspended in 0.5 ml buffer C (20mM HEPES, 25% glycerol, 0.42M
NaCl, 1.4mM MgCfr, 0.2mM EDTA, 0.5mM PMSF, 1 pg/ml leupeptin and
aprotinin, lOmM P-glycerophosphate).
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73
After incubation o f nuclear lysates at 4 ° C on a rocker for 30 minute, nuclear
proteins were obtained by centrifugation at 4 °C for 30 minutes at 13000 rpm. Both
cytoplasmic and nuclear protein extracts were stored at -80 0 C.
Two-dimensional gel electrophoresis
Isoelectric focusing was carried out in glass tubes of inner diameter 2 mm
using 2% pH 3.5-10 Ampholines (Amersham Pharmacia Biotech, Piscataway, NJ)
for 9600 volt-hrs. 1 jig oflEF internal standard, tropomyosin, was added to each
sample. This protein migrates as a doublet with lower polypeptide spot o f MW
33,000 and pi 5.2. After equilibration for 10 minutes in buffer ‘O’ (10% glycerol,
50mM dithiothreitol, 2.3% SDS and 0.0625 M Tris, pH 6.8) the tubes were sealed at
the top of stacking gels on top o f 10% acrylamide slab gels. SDS slab gel
electrophoresis was carried out for about 4 hours at 12.5 mA/gel. After slab gel
electrophoresis the gel was transblotted onto PVDF membrane in transfer buffer
(12.5mM Tris, pH 8.8, 86mM Glycine, 10% Methanol) overnight at 200mA and
approximately 100 volts/two gels.
Immunoprecipitation and Immunoblotting analyses
Antibodies used were anti-SUMO-1 (FL-101, Santa Cruz) polyclonal and
anti-GRP78 (C-20, Santa Cruz) monoclonal antibodies.
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74
Immunoprecipitation was performed with 400 pg cellular proteins and 2 pg primary
antibodies, which were incubated in 1 ml lysis buffer at 4 0 C for 2 hours. The 50 pi
of resuspended volume of protein A or G agarose conjugate were added to the above
reaction and incubated at 4 0 C overnight. Immunoprecipitates were collected by
centrifugation at 2500 rpm for 5 minutes at 4 0 C. The supernatants were discarded
and pellets were washed 4 times with 1 ml lysis buffer. After the final wash, the
supernatant was removed and the pellet was resuspend in 20 pi of 2X electrophoresis
sample buffer. The samples were boiled for 5 minutes and analyzed by SDS-PAGE.
Immunoblotting was performed with overnight electrotransfer to a PVDF membrane
(Bio-Rad) in TBST blocking buffer with 5 % non-fat dry milk. ECL reagent and
ECL-Hyperfilm (Amersham Pharmacia Biotech, Piscataway, NJ) were used for
detection.
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RESULTS AND DISCUSSION
75
GRP78 is sumoylated in ML-10 cells but this sumoylation disappeared in
MCV-50 clone.
As one of the ubiquitin-like proteins, sumo modifies important cellular
regulators. It does not mediate protein degradation, but does affect localization and
protein-protein interaction of substrates. Nuclear proteins of ML-10 and MCV-50
cells were elctrophoresed in two-dimensional gel followed by immunoblotting with
an antibody against sumo-1 (Fig. 8A, B). A sumoylated protein o f approximately 78
kDa was present in ML-10 cells but was lost in MCV-50 cells. Mass-spectrometry
analysis identified it as 78 kDa glucose regulated protein (GRP 78), and this was
confirmed by immuno-precipitation with antibody against SUMO followed by
immunoblotting with antibody against GRP 78 (Fig. 8C).
Although we didn’t investigate the above observation extensively, this
finding will help us to understand how sumoylation modulates GRP78 as well as
affects ovarian tumor progression.
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76
Figure 8. Differential sumoylation o f GRP78 in ML-10 and MCv-50. Nuclear
extracts from ML-10 cells (A) and MCV-50 cells (B) were electrophoresed on two-
dimensional gels and immunoblotted with antibody against SUMO. Arrow indicates
sumoylated protein (GRP78) that is lost in spontaneously immortalized MCV-50 cell
line. Arrowhead indicates internal standard, tropomyosin that migrates as protein
with 33 kDa and pi 5.2. GRP78 is a 78 kDa protein with pi 5.2. Immunoprecipitation
with antibody against SUMO followed by immunoblotting with antibody against
GRP78 confirmed the identity of the sumoylated protein is GRP78 (C). N. C. is a
negative control for immuno-precipitation that does not contain any protein extract.
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A
7 7
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CHAPTER IV
78
CONCLUSIONS AND FUTURE DIRECTIONS
At the beginning of this study, I sought to see if there was significant change
in ubiquitylation o f cellular proteins in our in vitro longitudinal model of ovarian
tumor progression. This model was developed by introducing an adenoviral vector
containing SV40 large T antigen into primary explanted cultures o f ovarian
cystadenomas. The resulting mortal cell strain called ML-10 further gave rise to two
spontaneously immortalized clones called MCV-39 and MCV-50. Immunoblotting
o f nuclear proteins extracted from those cells with anti-ubiquitin antibody revealed
that at least three distinct proteins were differentially ubiquitylated. Two of these
proteins were upregulated and the other was downregulated in spontaneoulsly
immortalized MCV-39 and MCV-50 clones. One o f these upregulated proteins was
identified as human nucleolin by immunoprecipitation followed by amino acid
sequence analysis. Although human nucleolin is composed of 707 amino acids, it
behaves as a 105 kDa protein in SDS-PAGE due to its acidic N-terminal domain.
Since one ubiquitin molecule has a mass of 8.5 kDa and the ubiquitylated nucleolin
appears as single band with an apparent molecular weight close to 105 kDa, I assume
that nucleolin is monoubiquitylated.
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79
Studying the significance o f this novel finding should increase our understanding of
the biological function of nucleolin as well as of its regulation. Further studies on the
relationship between monoubiquitylation and other post-translational modifications
such as phosphorylation and methylation should be particularity informative.
Although nucleolin methylation was not examined in this thesis, I showed
evidence of association between nucleolin ubiquitylation and phosphorylation.
Monoubiquitylated nucleolin was predominant in unphophorylated fraction of
intracellular proteins. All cytoplasmic monoubiquitylated nucleolins was
phosphorylated while nuclear monoubiquitylated nucleolin was either
phosphorylated or unphosphorylated. Based on the above observation, we
hypothesize that nucleolin with both modifications acquires the ability to
translocalize to the cytoplasm where it is further cleaved.
To validate the above hypothesis, a number o f questions need to be answered.
First is which modification can modulate the other. In cell cycle and transcriptional
regulation, phosphorylation and ubiquitylation work in an orderly manner to
modulate the function of regulatory proteins. Knowledge of the exact site of
monoubiquitylation should facilitate answer the above question. This goal may be
achieved by mass-spectrometry comparison o f GST-Nucleolin fusion protein lacking
the monoubiquitylation due to its expression in bacteria and of the mono
ubiquitylated nucleolin from ovarian carcinoma cells.
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80
Classical deletion and point mutation assays may not be useful because lysine
residues that broadly locate from N-terminal to C-terminal end of nucleolin comprise
more than 12 % o f total amino acids and the transfection and over-expression of
nucleolin in cells was problematic due to its toxicity. As alternative approach, point
mutations of already known phosphorylation sites may reveal which modification
depends on the other modification as well as which phosphorylation is associated
with the monoubiquitylation o f nucleolin. GST-Nucleolin is invaluable because it
makes it possible not only to locate a specific Lys residue responsible for the
monoubiquitylation, but also to investigate the above questions through in vitro
ubiquitylation and phosphorylation. GST-Nucleolin with point mutation of specific
phosphorylation or ubiquitylation residues may be a substrate for in vitro
ubiquitylation or phosphorylation, which may elucidate cross regulation between two
post-translational modifications. Given that ubiquitylation is an enzymatic post-
translational modification o f proteins, the identification of proteins involved in the
monoubiquitylation is important to further our understanding of this process. GST-
Nucleolin can be used as bait for in vitro ubiquitylation and phosphorylation assay to
pull down specific proteins that are associated with both modifications.
A second question is whether nucleo-cytoplasmic transport of
monoubiquitylated nucleolin is uni-directional. It is important to know which cellular
compartments and which enzymes are responsible for both modifications of
nucleolin.
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81
If the enzyme responsible for the monoubiquitylation o f nucleolin exists only in
nucleus, nucleolin with both modifications found in cytoplasm may be exported from
nucleus. The observation that nucleolin with both modifications is the only modified
form found in the cytoplasm implies that these modifications may occur in the
nucleus and that nucleo-cytoplasmic transport of modified nucleolin might be uni
directional. However, it can be ruled out that nucleolin could be constantly shuttling,
and both modifications could block the re-entry o f nucleolin into nucleus.
A third question regards the identity o f the monoubiquitylated and
phosphorylated 80 kDa nucleolin fragment that is only found in the cytoplasm. It has
been suggested that the stability of nucleolin depends on the level of phosphorylation
since the phosphorylation o f nucleolin might enhance its auto-degradation by
proteases (193). This fragmented nucleolin with both modifications may have a
specific function or be an initiative form for further degradation.
The glucose-regulated protein GRP78 functions as a molecular chaperone.
It is expressed constitutively in the endoplasmic reticulum in most cell types under
normal growth conditions and is highly induced in stressed cells. This protein is
essential for the proper glycosylation, folding and assembly o f many membrane
bound and secreted proteins (225). Inducing factors are cellular environments of
low glucose or oxygen and reagents that disrupt the ER function such as calcium
ionophores (226).
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82
GRP78 is critical for maintenance of cell homeostasis and anti-apoptosis
(227). We observed that GRP78 is sumoylated in mortal ML-10 cells but not in
spontaneously immortalized daughter MCV-50 cells. The molecular mechanism as
well as functional significance o f the sumoylated GRP78 is another interesting
subject for future investigations.
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Creator Shin, Bongha (author)

Core Title Post-translational modification of nucleolin by monoubiquitylation in human ovarian carcinoma cells

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Pathobiology

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

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

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-333787

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