Abstract 3077: The SR protein kinase Clk1 phosphorylates SPF45 and regulates its localization, degradation and alternative splicing activity

Abstract

Abstract Background: Alternative splicing (AS) of pre-mRNA is a major source of protein diversity in eukaryotic proteomes. Alterations in splicing factor expression have been shown in numerous cancers and reversible phosphorylation of splicing factors by SR protein kinases can regulate their localization and activity. The AS factor SPF45 (Splicing Factor 45) has low expression in normal tissues, but is overexpressed in several cancers. SPF45 overexpression was reported to induce multidrug resistance. There are no reports on how SPF45 activity is regulated by phosphorylation. Methods: To determine if Clk1 (Cdc2-like kinase 1) regulates SPF45 alternative splicing activity, we generated a delta-fas minigene and cotransfected COS-1 cells with delta-fas, wild-type or mutant SPF45, and active or kinase dead Clk1. Clk1 and SPF45 effects on delta-fas splicing were determined by RT-PCR. Clk1 inhibition was achieved by the chemical inhibitor TG003 or by Clk1 siRNA. Half life of endogenous, transiently-transfected and stably transfected SPF45 was determined by cycloheximide treatment and western blotting. Proteosome inhibition was achieved with MG132. Recombinant Clk1 was used to phosphorylate recombinant SPF45 in vitro. Phosphorylation sites were identified by (LC)-electrospray ionization (ESI) -tandem mass spectrometry (MS/MS) and confirmed by in vitro kinase assays. Confocal immunofluorescence was performed to determine the subnuclear localization of wild type and mutant SPF45 upon manipulation of Clk1 expression or activity. Results: Clk1 expression significantly increased SPF45-mediated delta-fas alternative splicing, while Clk1 inhibition with kinase-dead Clk1, Clk1 siRNA, or TG003 significantly inhibited SPF45 AS activity. Clk1 expression enhanced expression of SPF45 protein, but not mRNA, while Clk1 inhibition decreased SPF45 protein expression. Clk1 inhibition increased SPF45 degradation through a proteosome-independent pathway. Clk1 phosphorylated SPF45 in vitro on Ser202 and Ser204 and mutation of these residues partially inhibited SPF45 AS activity, although Clk1 still partially induced AS activity of a SPF45-S202/204 mutant, suggesting that the effects of Clk1 on SPF45 were both Ser202/204 dependent and independent. Confocal immunofluorescence studies demonstrated that expression of active Clk1 caused a redistribution of SPF45 from nuclear speckles to a diffuse pattern within the nucleus in a Ser202/Ser204 dependent manner, while inhibition of Clk1 blocked these effects. Conclusion: Clk1 is the first kinase demonstrated to phosphorylate SPF45, increasing its expression and alternative splicing activity while changing SPF45 subnuclear localization. These data provide new insights into the regulation of SPF45 and raise the possiblity that inhibition of Clk1 could be used as a therapeutic target for SPF45-mediated drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3077. doi:10.1158/1538-7445.AM2011-3077