Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.
Poster PresentationsP213 1, 2, 3, 4 kHz) the preoperative and postoperative air-bone gap were analyzed in order to ascertain whether the preoperative ABG could act as a predictor. Having not passed normality testing, Wilcoxon matched paired signed rank test and Spearman rank coefficient were used for analysis.Results: Throughout the statistical analysis, preoperative ABG correlated most strongly with net difference in postoperative ABG (P < .0001). For each commonly tested frequency assessed this demonstrated a "very strong" positive correlation. As such, the greater the preoperative ABG, the greater the closure obtained through stapedectomy in this series.Conclusions: In performing stapedectomy for otosclerosis, our series suggested the preoperative ABG may have a role in preoperative patient counseling and consent but is unable to predict success in stapedectomy surgery.
Src phosphorylates splicing factor 45 (SPF45) and regulates SPF45 nucleocytoplasmic shuttling, cell migration and invasion Yuying Liu1, LaShardai Conaway1, Jennifer Rutherford Bethard1, Amber Thompson Bradley1 and Scott T. Eblen1,2 From Department of Cell and Molecular Pharmacology and Experimental Therapeutics1 and the Hollings Cancer Center2 Medical University of South Carolina, Charleston, SC, 29425 SPF45 is a bifunctional protein involved in splicing and DNA repair and is overexpressed in several forms of cancer. Previously, we have shown that SPF45 overexpression regulates ovarian cancer cell proliferation and adhesion in a MAP kinase phosphorylation-dependent manner. In this study, we investigated novel roles of SPF45 and their regulation by the tyrosine kinase Src. We show by cell fractionation that endogenous SPF45 localizes in both the nucleus and cytoplasm and that exposure of cells to UV irradiation or osmotic shock increased SPF45 cytoplasmic accumulation. Consistent with this result, heterokaryon assays using SKOV-3 cells stably expressing Myc-SPF45 showed that SPF45 shuttles between the nucleus and cytoplasm and that pretreatment of the cells with the Src family kinase inhibitor PP2 inhibited nucleocytoplasmic shuttling. Src strongly induced SPF45 tyrosine phosphorylation and co-immunoprecipitated with SPF45 after co-transfection into COS-1 cells. Src phosphorylated SPF45 in vitro on five tyrosine residues (Y100, Y108, Y194, Y214 and Y246), as determined by mass spectrometry. Sequential mutation of these sites to phenylalanine inhibited SPF45 tyrosine phosphorylation in cells co-transfected with Src, with mutation of all 5 sites (Myc-SPF45-Y5F) completely inhibiting SPF45 tyrosine phosphorylation. Myc-SPF45-Y5F stably expressed in SKOV-3 cells showed reduced nucleocytoplasmic shuttling compared to wild type Myc-SPF45. SKOV-3-Myc-SPF45 cells demonstrated enhanced migration and invasion compared to vector control cells, while SKOV-3-Myc-SPF45-Y5F cells migrated significantly less than SKOV-3-Myc-SPF45 cells. These data are the first to show that SPF45 is a shuttling protein, a Src substrate, and that Src phosphorylation of SPF45 regulates nucleocytoplasmic shuttling and SPF45-induced cell migration and invasion. Citation Format: Yuying Liu, Lashardai Neniara Conaway, Jennifer Rutherford Bethard, Amber Thompson Bradley, Scott Eblen. Src phosphorylates splicing factor 45 (SPF45) and regulates SPF45 nucleocytoplasmic shuttling, cell migration and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4283. doi:10.1158/1538-7445.AM2013-4283
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.