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.

High temporal resolution of gene expression dynamics in developing mouse embryonic stem cells

“…Supporting the efficacy of the approach, we found Clock , which encodes a key regulator of circadian rhythm in mammals, to have a period of 24.2 hours. We identified 20 genes that displayed characteristics of both slRNAs and cycRNAs (Supplementary Figure S2F), including Ewsr1 and Clk1 , involved in gene splicing(Paronetto et al 2011; Liu et al 2013) as well as five uncharacterized lncRNAs. Given the highly specific expression patterns in this context, we propose these genes may similarly have roles in maintaining or establishing biological rhythms.…”

Extracellular-Regulated Kinases: Signaling From Ras to ERK Substrates to Control Biological Outcomes

“…M. Al-Ayoubi et al, 2012). EDA inclusion occurs less in adult tissues, but is mainly seen in embryonic tissue, fibrotic liver, and wound healing (Blaustein, Pelisch, & Srebrow, 2007), and we have shown that SPF45 overexpression enhances cell migration (Y. Liu et al, 2013).…”

Connecting the speckles: Splicing kinases and their role in tumorigenesis and treatment response

“…99,100 A recent study has shown that CLK1 directly phosphorylates SPF45 on 8 serine residues, and that this phosphorylation led to the stabilization of SPF45 protein levels, and regulated exon 6 skipping in Fas pre-mRNA. 101 Furthermore, SPF45 overexpression induced cell migration and invasion in ovarian cancer cells, 101 suggesting CLK1 mediated stabilization of SPF45 could impact multiple aspects of tumor progression.…”