Mitogen-Activated Protein Kinase Phosphorylation of Splicing Factor 45 (SPF45) Regulates SPF45 Alternative Splicing Site Utilization, Proliferation, and Cell Adhesion

Abstract

The regulation of alternative mRNA splicing factors by extracellular cues and signal transduction cascades is poorly understood. Using an engineered extracellular signal-regulated kinase 2 (ERK2) that can utilize ATP analogs, we have identified the alternative mRNA splicing factor 45 (SPF45), which is overexpressed in cancer, as a novel coimmunoprecipitating ERK2 substrate. ERK2 phosphorylated SPF45 on Thr71 and Ser222 in vitro and in cells in response to H-RasV12, B-RAF-V600E, and activated MEK1. Jun N-terminal kinase 1 (JNK1) and p38␣ also phosphorylated SPF45 in vitro and associated with SPF45 in cells. SPF45 was differentially phosphorylated in cells by all three mitogen-activated protein (MAP) kinases in response to phorbol myristate acid (PMA), H 2 O 2 , UV, and anisomycin stimulation. ERK and p38 activation decreased SPF45-dependent exon 6 exclusion from fas mRNA in a minigene assay in cells. Stable overexpression of SPF45 in SKOV-3 cells dramatically inhibited cell proliferation in a phosphorylation-dependent manner through inhibition of ErbB2 expression. SPF45 overexpression also induced EDA inclusion into fibronectin transcripts and fibronectin expression in a phosphorylation-dependent and -independent manner, respectively, specifically affecting cellular adhesion to a fibronectin matrix. These data identify SPF45 as the first splicing factor regulated by multiple MAP kinase pathways and show effects of both SPF45 overexpression and phosphorylation.T he expression of more than one protein from a single gene is regulated by alternative mRNA splicing, in which the exons from pre-mRNA of a transcribed gene are differentially spliced together (6), affecting the composition of the final protein product. Alternative splicing is thought to regulate between 60 and 74% of the human genome (42, 66), and up to 50% of human genetic diseases arise from changes in alternative splicing (38). Pre-mRNA splicing is regulated by both small nuclear ribonucleoprotein particles (snRNPs) and proteins that function in the stepwise processing of pre-mRNA (29, 65). Alternative splicing is primarily regulated by the hnRNP (heterogenous nuclear ribonucleoproteins) and SR (serine-arginine-rich) families of splicing factor proteins (28,37,41). Other alternative splicing factors fall outside these families and contain one or more RNA recognition motifs (RRMs) and protein-protein binding domains. Splice site selection depends on the relative concentrations of these proteins (8,27) and is regulated by reversible phosphorylation (57). Little is known about how extracellular signals and intracellular signal transduction regulate pre-mRNA splicing.The alternative mRNA splicing factor SPF45 (splicing factor 45) was identified in mass spectrometry analysis of the spliceosome complex (45) and acts in the second step of splicing, regulating 3= recognition of alternative splice sites in pre-mRNA of the sxl gene in Drosophila (33). SPF45 regulates alternative splicing of pre-mRNA encoding the death receptor fas in minigene assays in cells, ind...

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