title: Mechanism of dephosphorylation of the SR protein ASF/SF2 by protein phosphatase 1.
abstract: SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, we monitored region-specific mapping of phosphorylation sites in ASF/SF2 as a function of the protein phosphatase PP1. We showed that 10 phosphates added to the RS1 segment by SRPK1 are removed in a preferred N-to-C manner, directly opposing the C-to-N phosphorylation by SRPK1. Two N-terminal RNA recognition motifs in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RNA recognition motifs protects against dephosphorylation, suggesting that engagement of the SR protein with exonic splicing enhancers can regulate phosphoryl content in the RS domain. In addition to regulation by N-terminal domains, phosphorylation of the C-terminal portion of the RS domain (RS2) by the nuclear protein kinase Clk/Sty inhibits RS1 dephosphorylation and disrupts the directional mechanism. The data indicate that both RNA-protein interactions and phosphorylation in flanking sequences induce conformations of ASF/SF2 that increase the lifetime of phosphates in the RS domain.
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: 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: D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors
chemical_list: D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D000068103:Serine-Arginine Splicing Factors; D012313:RNA; C066780:Clk dual-specificity kinases; C087728:SRPK1 protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054645:Protein Phosphatase 1
references.citation: Mol Cell. 2003 Jul;12(1):5-14
references.pmid: 12887888
references.citation: J Mol Biol. 1995 Dec 15;254(5):942-59
references.pmid: 7500362
references.citation: J Cell Biol. 1999 May 3;145(3):447-55
references.pmid: 10225947
references.citation: J Mol Biol. 2001 Mar 9;306(5):997-1010
references.pmid: 11237614
references.citation: Mol Cell. 2005 Dec 22;20(6):881-90
references.pmid: 16364913
references.citation: Mol Cell Biol. 1999 Oct;19(10):6991-7000
references.pmid: 10490636
references.citation: EMBO J. 1996 Jan 15;15(2):265-75
references.pmid: 8617202
references.citation: FEBS Lett. 1992 Jul 13;306(1):17-22
references.pmid: 1628739
references.citation: J Biol Chem. 2006 Jul 28;281(30):20689-20697
references.pmid: 16707485
references.citation: Biochemistry. 2009 Dec 8;48(48):11432-40
references.pmid: 19886675
references.citation: Hum Mol Genet. 2008 Jan 1;17(1):52-70
references.pmid: 17913700
references.citation: Mol Cell. 2008 Mar 14;29(5):563-76
references.pmid: 18342604
references.citation: Proc Natl Acad Sci U S A. 1999 Sep 14;96(19):10655-60
references.pmid: 10485881
references.citation: Cell. 1993 Dec 17;75(6):1061-70
references.pmid: 8261509
references.citation: RNA. 1995 Sep;1(7):692-706
references.pmid: 7585254
references.citation: J Biol Chem. 1999 Apr 16;274(16):11125-31
references.pmid: 10196197
references.citation: Nucleic Acids Res. 1992 Oct 25;20(20):5263-9
references.pmid: 1331983
references.citation: Mol Biol Cell. 1992 Mar;3(3):299-308
references.pmid: 1627831
references.citation: Nature. 1994 Mar 10;368(6467):119-24
references.pmid: 8139654
references.citation: Mol Cell Biol. 2002 Oct;22(19):6871-82
references.pmid: 12215544
references.citation: Exp Cell Res. 1998 Jun 15;241(2):300-8
references.pmid: 9637771
references.citation: J Biol Chem. 2001 Aug 24;276(34):32382-91
references.pmid: 11432864
references.citation: J Mol Biol. 2008 Oct 17;382(4):894-909
references.pmid: 18687337
references.citation: J Mol Biol. 2008 Feb 8;376(1):55-68
references.pmid: 18155240
references.citation: J Mol Biol. 2009 Jul 24;390(4):618-34
references.pmid: 19477182
references.citation: Biochem Cell Biol. 1999;77(4):293-8
references.pmid: 10546892
references.citation: J Biol Chem. 1997 Jul 25;272(30):19008-16
references.pmid: 9228083
references.citation: J Biol Chem. 2005 Dec 16;280(50):41761-8
references.pmid: 16223727
references.citation: Mol Cell. 2005 Oct 7;20(1):77-89
references.pmid: 16209947
references.citation: EMBO J. 1994 Dec 1;13(23):5679-88
references.pmid: 7988565
references.citation: Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):12601-6
references.pmid: 14555757
references.citation: J Biol Chem. 2007 Sep 28;282(39):28874-28883
references.pmid: 17636256
references.citation: Trends Biochem Sci. 2000 Mar;25(3):106-10
references.pmid: 10694877