Defense Date

2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Dr. Steven R. Grossman

Second Advisor

Dr. Larisa Litovchick

Third Advisor

Dr. Sumitra Deb

Fourth Advisor

Dr. Kristoffer Valerie

Fifth Advisor

Dr. Joseph Landry

Abstract

CBP (CREB Binding Protein) is global transcriptional co-activator and histone acetyltransferase. CBP is involved in the modulation of the transcription of many genes via histone acetylation at the promoter regions of the target genes. Also, non-histone proteins and transcription factors can be acetylated by CBP to promote their transcriptional activation. In addition to its transcription co-activator role, CBP is involved in many other pathological and physiological cellular processes such as cell growth and differentiation, cell transformation and development, response to stress, cell cycle regulation and apoptosis.

CBP and its paralogue p300, play double-edged roles in the regulation of p53, a well characterized tumor suppressor protein, via ubiquitination and acetylation activities. Prior work has shown that CBP and p300 contribute to the maintenance of physiologic p53 levels in unstressed cells via a cytoplasmic but not nuclear, p53-directed E4 polyubiquitin ligase activity, subsequently leading to p53 proteasomal degradation. Our previous work also revealed that CBP and p300 possess intrinsic cytoplasmic but not nuclear E3 autoubiquitination activity in the absence of cellular stress. The mechanism of the compartmentalized CBP/p300 ubiquitin ligase activities was not studied. In this thesis, I present insights gained from efforts to determine the regulation of CBP ubiquitin ligase activities in the cytoplasm versus nucleus, in the absence and presence of DNA damage stress. Chapter two discusses the effect of DNA damage on CBP E3 autoubiquitination activity and also addresses the differential post translational modifications between cytoplasmic and nuclear CBP, in the absence and presence of DNA damage. Aspects of the regulation of the compartmentalized CBP ubiquitin ligase activities in the absence of cellular stress were covered in chapter three. We employed Multidimensional Protein Identification Technology (MudPIT) and mass spectrometry analysis of purified cytoplasmic and nuclear CBP to identify nuclear and cytoplasmic CBP interacting proteins. MudPIT analysis revealed that Cell Cycle and Apoptosis Regulator protein (CCAR2), also known as Deleted in Breast Cancer 1 protein (DBC1), is a novel CBP-interacting protein, in the cytoplasmic and nuclear compartments. Functional analysis suggested that DBC1 directly regulates cellular compartmentalization of CBP E3 and p53-directed E4 ubiquitination activities. This work identifies the different regulatory mechanisms of differential CBP ubiquitin ligase activities in the absence and presence of DNA damage. Remarkably, DBC1 was identified as a novel binding partner of CBP and a critical regulator of CBP ubiquitination activities towards p53.This work may provide novel strategies for the development of cancer therapeutics against tumors maintaining wild type p53, which have deleted DBC1.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-10-2016

Available for download on Saturday, August 07, 2021

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