Date of Award

2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

College of Graduate Studies

First Advisor

Scott T. Eblen

Second Advisor

Kenneth D. Tew

Third Advisor

Steven A. Rosenzweig

Fourth Advisor

Bryan Toole

Fifth Advisor

Dennis K. Watson

Abstract

Alternative pre-mRNA splicing increases proteome diversity and is important in the behavior of cells in health and disease. The processes that regulate alternative premRNA splicing are diverse and include regulation of splicing factors by phosphorylation. Here we report that the pre-mRNA alternative splicing factor SPF45 is a novel substrate of ERK, JNK and p38 MAP kinases in ovarian cancer cells. Using mutational analysis and phospho-specific antibodies, we demonstrate that MAP kinases phosphorylate SPF45 on Thr71 and Ser222 and that phosphorylation in cells is induced by a number of extracellular stimuli including PMA, EGF, serum, H20 2 and UV. Exon 6 of the death receptor Fas/CD95 has been shown to undergo alternative splicing in the presence of SPF45. Using a Fas minigene assay, we show that ERK2 and p38 inhibit SPF45 alternative splicing activity towards Fas, dependent upon these two phosphorylation sites. Other than Fas, no other pre-mRNA targets of SPF45 have been reported in mammalian cells. We generated SKOV3 cells stably-overexpressing wild-type SPF45 or a phosphorylation site mutant and performed an exon and gene array analysis to identify novel SPF45 splicing targets and genes whose expression is changed downstream of SPF45 splicing activity, respectively. From this analysis, 139 potential splicing targets and over 150 genes with altered expression were identified. We focus on four genes for validation with emphasis on ErbB2 and fibronectin. We show that SPF45 downregulates cellular proliferation in SKOV3 cells in a phosphorylation-dependent manner. Furthermore, we demonstrate that SPF45 regulates fibronectin alternative splicing, enhances inclusion of FN-EDIIIA region, and affects cellular adhesion to fibronectin matrix. We also assess SPF45 binding to SFl and SF3b15S, essential components of the spliceosome, as well as the impact of Thr71 and Ser222 mutations on this interaction. Finally, we determine the effect of SPF45 overexpression in SKOV3 cells on their drug resistance profile. This study provides a link between MAP kinase signaling and splicing factors and identifies the role of this interaction in regulating molecular processes in ovarian cancer cells. Additionally, it provides the basis to investigate the role of SPF45 in ovarian cancer and to produce successful therapeutic interventions targeting SPF45-mediated effects.

Rights

All rights reserved. Copyright is held by the author.

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