title: Comprehensive analysis of phosphorylation sites in Tensin1 reveals regulation by p38MAPK.
abstract: Tensin1 is the archetype of a family of focal adhesion proteins. Tensin1 has a phosphotyrosine binding domain that binds the cytoplasmic tail of β-integrin, a Src homology 2 domain that binds focal adhesion kinase, p130Cas, and the RhoGAP called deleted in liver cancer-1, a phosphatase and tensin homology domain that binds protein phosphatase-1α and other regions that bind F-actin. The association between tensin1 and these partners affects cell polarization, migration, and invasion. In this study we analyzed the phosphorylation of human S-tag-tensin1 expressed in HEK293 cells by mass spectrometry. Peptides covering >90% of the sequence initially revealed 50 phosphorylated serine/phosphorylated threonine (pSer/pThr) but no phosphorylated tyrosine (pTyr) sites. Addition of peroxyvanadate to cells to inhibit protein tyrosine phosphatases exposed 10 pTyr sites and addition of calyculin A to cells to inhibit protein phosphatases type 1 and 2A gave a total of 62 pSer/pThr sites. We also characterized two sites modified by O-linked N-acetylglucosamine. Tensin1 F302A, which does not bind protein phosphatase-1, showed > twofold enhanced phosphorylation of seven sites. The majority of pSer/pThr have adjacent proline (Pro) residues and we show endogenous p38 mitogen activated protein kinase (MAPK) associated with and phosphorylated tensin1 in an in vitro kinase assay. Recombinant p38α MAPK also phosphorylated S-tag-tensin1, resulting in decreased binding with deleted in liver cancer-1. Activation of p38 MAPK in cells by sorbitol-induced hyperosmotic stress increased phosphorylation of S-tag-tensin1, which reduced binding to deleted in liver cancer-1 and increased binding to endogenous pTyr proteins, including p130Cas and focal adhesion kinase. These data demonstrate that tensin1 is extensively phosphorylated on Ser/Thr residues in cells and phosphorylation by p38 MAPK regulates the specificity of the tensin1 Src homology 2 domain for binding to different proteins. Tensin1 provides a hub for connecting signaling pathways involving p38 MAP kinase, tyrosine kinases and RhoGTPases.
authors.affiliation: Center for Cell Signaling, University of Virginia, 1400 Jefferson Park Ave, P.O. Box 800577-MSB7125, Charlottesville, VA 22908, USA.
authors.forename: Emily H
authors.initials: EH
authors.lastname: Hall
authors.forename: Jeremy L
authors.initials: JL
authors.lastname: Balsbaugh
authors.forename: Kristie L
authors.initials: KL
authors.lastname: Rose
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.forename: David L
authors.initials: DL
authors.lastname: Brautigan
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D053719:Tandem Mass Spectrometry; D000070876:Tensins; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D008840:Microfilament Proteins; C000603369:TNS1 protein, human; D000070876:Tensins; D048051:p38 Mitogen-Activated Protein Kinases
references.citation: Exp Biol Med (Maywood). 2002 Jun;227(6):412-24
references.pmid: 12037131
references.citation: Development. 2003 Sep;130(17):4001-10
references.pmid: 12874122
references.citation: J Cell Sci. 1999 Jun;112 ( Pt 11):1655-69
references.pmid: 10318759
references.citation: Anal Chem. 2004 Jul 1;76(13):3590-8
references.pmid: 15228329
references.citation: J Biol Chem. 2009 Dec 11;284(50):34713-22
references.pmid: 19826001
references.citation: Oncogene. 1995 Sep 21;11(6):1149-55
references.pmid: 7566975
references.citation: Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9528-33
references.pmid: 15210983
references.citation: J Clin Invest. 1999 Mar;103(6):789-97
references.pmid: 10079099
references.citation: J Biol Chem. 2004 Sep 10;279(37):38466-70
references.pmid: 15247246
references.citation: Nat Protoc. 2008;3(11):1709-17
references.pmid: 18927556
references.citation: Mol Cell Biol. 2007 Jun;27(12):4217-27
references.pmid: 17438131
references.citation: Biochem J. 2005 Oct 15;391(Pt 2):359-70
references.pmid: 15975092
references.citation: Biochem Biophys Res Commun. 2000 Jun 16;272(3):717-20
references.pmid: 10860821
references.citation: ACS Chem Biol. 2007 Feb 20;2(2):109-18
references.pmid: 17256997
references.citation: J Cell Biol. 2007 Jan 1;176(1):43-9
references.pmid: 17190795
references.citation: J Cell Biol. 1994 Jun;125(5):1067-75
references.pmid: 8195290
references.citation: Int J Biochem Cell Biol. 2008;40(5):843-7
references.pmid: 17521951
references.citation: J Biol Chem. 2007 Jun 15;282(24):17806-15
references.pmid: 17435217
references.citation: Proc Natl Acad Sci U S A. 2006 Oct 3;103(40):14889-94
references.pmid: 17001009
references.citation: J Biol Chem. 2008 Sep 5;283(36):24949-61
references.pmid: 18586681
references.citation: J Biol Chem. 1996 Jun 7;271(23):13649-55
references.pmid: 8662921
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13793-8
references.pmid: 18779572
references.citation: Nat Biotechnol. 2008 Jan;26(1):127-32
references.pmid: 18183025
references.citation: Int J Biochem Cell Biol. 2004 Jan;36(1):31-4
references.pmid: 14592531
references.citation: J Biol Chem. 2003 Mar 28;278(13):11579-89
references.pmid: 12509440
references.citation: Biochem J. 2000 Oct 15;351 Pt 2:403-11
references.pmid: 11023826
references.citation: J Biol Chem. 1994 Sep 2;269(35):22310-9
references.pmid: 8071358
references.citation: Science. 1991 May 3;252(5006):712-5
references.pmid: 1708917
references.citation: Surgery. 1996 Aug;120(2):427-31; discussion 432
references.pmid: 8751614
references.citation: Proc Natl Acad Sci U S A. 2007 May 22;104(21):9012-7
references.pmid: 17517630
references.citation: J Biol Chem. 1996 Sep 20;271(38):23452-7
references.pmid: 8798552
references.citation: Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2272-7
references.pmid: 12606711
references.citation: J Biol Chem. 1993 Jul 15;268(20):14565-7
references.pmid: 8325835
references.citation: Neoplasia. 2004 Jan-Feb;6(1):1-6
references.pmid: 15068665