title: 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 p38α 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.
authors.affiliation: Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina, USA.
authors.forename: Adnan M
authors.initials: AM
authors.lastname: Al-Ayoubi
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
authors.forename: Yuying
authors.initials: Y
authors.lastname: Liu
authors.forename: Tao
authors.initials: T
authors.lastname: Bai
authors.forename: Scott T
authors.initials: ST
authors.lastname: Eblen
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion; D002460:Cell Line; D049109:Cell Proliferation; D017931:DNA Primers / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048308:Mitogen-Activated Protein Kinase 14 / Q000378:metabolism; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism; D010766:Phosphorylation; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D019014:fas Receptor / Q000235:genetics
chemical_list: D017931:DNA Primers; D048049:Extracellular Signal-Regulated MAP Kinases; D005353:Fibronectins; D019950:Mitogen-Activated Protein Kinase 1; D048308:Mitogen-Activated Protein Kinase 14; D048055:Mitogen-Activated Protein Kinase 8; D012322:RNA Precursors; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D019014:fas Receptor; C491901:FAS protein, human; C121693:RBM17 protein, human; C508053:ERBB2 protein, human; C535150:MAPK1 protein, human