title: Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion.
abstract: 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.
authors.affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425, USA.
authors.forename: Yuying
authors.initials: Y
authors.lastname: Liu
authors.forename: LaShardai
authors.initials: L
authors.lastname: Conaway
authors.forename: Jennifer
authors.initials: J
authors.lastname: Rutherford Bethard
authors.forename: Adnan M
authors.initials: AM
authors.lastname: Al-Ayoubi
authors.forename: Amber
authors.initials: A
authors.lastname: Thompson Bradley
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
authors.forename: Scott A
authors.initials: SA
authors.lastname: Weed
authors.forename: Scott T
authors.initials: ST
authors.lastname: Eblen
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement; D051356:Cortactin / Q000378:metabolism; D005091:Exons; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism
chemical_list: D051356:Cortactin; D005353:Fibronectins; C121693:RBM17 protein, human; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D019014:fas Receptor; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex