Publication Date

2014-02-05

Availability

Embargoed

Embargo Period

2015-02-05

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2014-01-29

First Committee Member

Joyce M. Slingerland

Second Committee Member

Zafar Nawaz

Third Committee Member

Thomas K Harris

Fourth Committee Member

Feng Gong

Fifth Committee Member

John Cleveland

Abstract

Many transcription factors undergo transcription-coupled proteolysis. While ligand binding activates ubiquitin-proteolysis of estrogen receptor alpha (ER), mechanisms governing this and its relationship to transcriptional activation were unclear. In this thesis, I present the evidence indicating that different ubiquitin ligases, including E6-AP and SCF/SKP2 regulate ER protein stability and transcriptional activity. SKP2 project support a model in which estrogen-activated CyclinE-CDK2 binds and phosphorylates ER-S341, to prime ER-SCF/SKP2 binding via SKP2-L248QTLL252 in late G1. SKP2 activates ER ubiquitylation and proteolysis. Putative late ER targets were identified by expression profiling, SKP2 knockdown attenuated E2F-1 and BLM induction. SKP2 overexpression, but not coactivator motif mutant SKP2-L248QTAA252, enhanced estrogen-induced E2F-1 and BLM expression. SKP2 knockdown impaired estrogen-stimulated ER, SKP2, SRC3 and PolII recruitment to E2F-1 and BLM promoters. This work not only identifies these late-activated genes as bone fide ER targets but describes a novel mechanism for their periodic activation. SKP2 serves as dual ER E3 ligase/coactivator for late-activated target genes, revealing a novel mechanism whereby ER:SCF/SKP2 transactivation of E2F-1 feeds forward to drive G1-to-S. Experimental work of this thesis has elucidated novel molecular mechanisms whereby E3 ubiquitin ligases play dual roles as ER coactivators. These studies link ER proteolysis and activation of ER target genes and may not only prove to be relevant to hormone regulation of breast cancer but also reveal mechanisms whereby low level receptor expression governs gene action in several normal target tissues.

Keywords

E3 ligase; E6-AP; Estrogen receptor; FBXL6; SKP2; Src

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