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POST-TRANSLATIONAL MODIFICATION CROSSTALK
REGULATES KAP1 CO-REPRESSOR FUNCTIONS IN RESPONSE TO
DNA DAMAGES
by
Xu Li
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
December 2009
Copyright 2009 Xu Li
Object Description
| Title | Post-translational modification crosstalk regulates KAP1 co-repressor functions in response to DNA damages |
| Author | Li, Xu |
| Author email | xuli@usc.edu; xl@coh.org |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Molecular Pharmacology & Toxicology |
| School | School of Pharmacy |
| Date defended/completed | 2009-10-20 |
| Date submitted | 2009 |
| Restricted until | Unrestricted |
| Date published | 2009-11-28 |
| Advisor (committee chair) |
Shen, Wei-Chiang Ann, David K. |
| Advisor (committee member) |
Okamoto, Curtis Stallcup, Michael Stiles, Bangyan |
| Abstract | As a multifunctional protein, KRAB domain-associated protein 1 (KAP1) is reportedly subjected to multiple protein post-translational modifications, including phosphorylation and SUMOylation. However, gaps exist in our knowledge of how KAP1 phosphorylation crosstalks with KAP1 SUMOylation, how those post-translational modifications are regulated and what the biological consequence is.; In the first part of study, we found that doxorubicin (Dox)-treatment induces KAP1 phosphorylation at Ser-824 in an ataxia telangiectasia mutated (ATM)-dependent manner, correlating with the transcriptional de-repression of p21WAF1/CIP1 and Gadd45α. A S824A substitution of KAP1, which ablates the ATM-induced phosphorylation, results in an increase of KAP1 SUMOylation and repression of p21 transcription in Dox-treated cells. By contrast, a S824D mutation of KAP1, which mimics constitutive phosphorylation of KAP1, leads to a decrease of KAP1 SUMOylation and stimulation of p21 transcription before the exposure of Dox. We further provided evidence that SENP1 deSUMOylase is involved in activating basal, but not Dox-induced, KAP1 Ser-824 phosphorylation, rendering a stimulation of p21WAF1/CIP1 and Gadd45α transcription by attenuating KAP1 basal SUMOylation. Moreover, KAP1 and differential SUMOylation of KAP1 were also demonstrated to fine-tune the transcription of three additional KAP1-targeted genes, including Bax, Puma, and Noxa. Taken together, our results suggest a novel role for ATM that selectively stimulates KAP1 Ser-824 phosphorylation to repress its SUMOylation, leading to the de-repression of expression of a subset of genes involved in promoting cell cycle control and apoptosis in response to genotoxic stresses.; In the second part of study, we found that while protein phosphatase PP1cA directly interacts with KAP1, myosin phosphatase targeting subunit 1 bridges the PP1cB-KAP1 interaction. PP1cA or PP1cB overexpression/knockdown leads to a differential inverse co-regulation of basal and DNA double-strand break (DSB)-induced KAP1 Ser-824-phosphorylation versus SUMOylation. ChIP-ReIP experiments revealed PP1cA and PP1cB were recruited to KAP1, with different kinetics pre and post DSB induction, providing a mechanistic basis for the KAP1 dephosphorylation-SUMOylation switch. Interestingly, PP1cB stimulated KAP1 SUMOylation by both Ser-824-dephosphorylation-dependent and -independent mechanisms. We posit a novel mechanism whereby PP1 confers KAP1 Ser-824-dephosphorylation and assures KAP1 SUMOylation to counter the ATM effect, regulating transcription of KAP1-targeted genes in unstressed and stressed cells.; In the third part of study, we observed a decrease in the steady-state level of KAP1 upon overexpression of SUMO-2 under Dox-treatment. By using KAP1(S824D) to mimic Dox-induced, ATM-mediated KAP1 Ser-824 phosphorylation, K554R mutation blocked SUMO-promoted degradation. By utilizing co-IP assay, we found that KAP1 is ubiquitinated in vivo and that SUMO-2 greatly enhances KAP1 ubiquitination. We then explored the possibility that RNF4 (really interesting new gene) mediates SUMO-dependent ubiquitination and degradation of KAP1. Our results suggested that RNF4 serves as the E3 ligase of KAP1 SUMO-dependent ubiquitination and degradation. These findings may explain how KAP1 phosphorylation antagonizes its SUMOylation. Our results presented in this chapter suggested that, KAP1 Ser-824 phosphorylation leads to the degradation of already SUMOylated KAP1. |
| Keyword | DNA damage responses; KAP1; PP1; RNF4; SUMOylation; phosphorylation; ubiquitination; post-translational modifications crosstalk; doxorubicin |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m2774 |
| Rights | Li, Xu |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-LI-3376 |
| Archival file | uscthesesreloadpub_Volume32/etd-LI-3376.pdf |
Description
| Title | Page 1 |
| Full text | POST-TRANSLATIONAL MODIFICATION CROSSTALK REGULATES KAP1 CO-REPRESSOR FUNCTIONS IN RESPONSE TO DNA DAMAGES by Xu Li A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MOLECULAR PHARMACOLOGY AND TOXICOLOGY) December 2009 Copyright 2009 Xu Li |
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