title: Ordered multi-site phosphorylation of the splicing factor ASF/SF2 by SRPK1.
abstract: The human alternative splicing factor ASF/SF2, an SR (serine-arginine-rich) protein involved in mRNA splicing control, is activated by the multisite phosphorylation of its C-terminal RS domain, a segment containing numerous arginine-serine dipeptide repeats. The protein kinase responsible for this modification, SR-specific protein kinase 1 (SRPK1), catalyzes the selective phosphorylation of approximately a dozen serines in only the N-terminal portion of the RS domain (RS1). To gain insights into the nature of selective phosphate incorporation in ASF/SF2, region-specific phosphorylation in the RS domain was monitored as a function of reaction progress. Arg-to-Lys mutations were made at several positions to produce unique protease cleavage sites that separate the RS domain into identifiable N- and C-terminal phosphopeptides upon treatment with lysyl endoproteinase. These studies reveal that SRPK1 docks near the C-terminus of the RS1 segment and then moves in an N-terminal direction along the RS domain. Multiple quadruple Ser-to-Ala and deletion mutations did not disrupt the phosphorylation of other sites regardless of position, suggesting that the active site of SRPK1 docks in a flexible manner at the center of the RS domain. Taken together, these data suggest that SRPK1 uses a unique 'grab-and-pull' mechanism to control the regiospecific phosphorylation of its protein substrate.
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, USA.
authors.forename: Chen-Ting
authors.initials: CT
authors.lastname: Ma
authors.forename: Adolfo
authors.initials: A
authors.lastname: Velazquez-Dones
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Hagopian
authors.forename: Gourisankar
authors.initials: G
authors.lastname: Ghosh
authors.forename: Xiang-Dong
authors.initials: XD
authors.lastname: Fu
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Adams
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D017384:Sequence Deletion; D000068103:Serine-Arginine Splicing Factors
chemical_list: D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D000255:Adenosine Triphosphate; C087728:SRPK1 protein, human; D017346:Protein Serine-Threonine Kinases