title: Serine dephosphorylation of receptor protein tyrosine phosphatase alpha in mitosis induces Src binding and activation.
abstract: Receptor protein tyrosine phosphatase alpha (RPTPalpha) is the mitotic activator of the protein tyrosine kinase Src. RPTPalpha serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPalpha Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPalpha pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPalpha, and intrinsic catalytic activity of RPTPalpha was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPalpha was induced in mitosis. GRB2 binding to RPTPalpha, which was proposed to compete with Src binding to RPTPalpha, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPalpha-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPalpha, illustrating that Src binding to RPTPalpha is not mediated by a pTyr-SH2 interaction. Mutation of RPTPalpha Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPalpha pSer204 facilitates Src binding, leading to RPTPalpha-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.
authors.affiliation: Hubrecht Institute-KNAW and University Medical Center, Utrecht, Netherlands.
authors.forename: Andrei M
authors.initials: AM
authors.lastname: Vacaru
authors.forename: Jeroen
authors.initials: J
authors.lastname: den Hertog
mesh_terms: D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D055162:Biocatalysis; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008938:Mitosis; D008954:Models, Biological; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011485:Protein Binding; D054648:Protein Phosphatase 2 / Q000378:metabolism; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4 / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D045163:Antibodies, Phospho-Specific; D010768:Phosphoserine; D019061:src-Family Kinases; D054648:Protein Phosphatase 2; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4
references.citation: EMBO J. 2000 Mar 1;19(5):964-78
references.pmid: 10698938
references.citation: J Biol Chem. 1995 Sep 29;270(39):23126-31
references.pmid: 7559456
references.citation: EMBO J. 2002 Feb 15;21(4):493-503
references.pmid: 11847098
references.citation: Biochem J. 1997 Nov 1;327 ( Pt 3):867-76
references.pmid: 9581568
references.citation: J Biol Chem. 2002 Jun 14;277(24):21922-9
references.pmid: 11923305
references.citation: Mol Pharmacol. 2005 Apr;67(4):1206-13
references.pmid: 15630078
references.citation: FEBS Lett. 1997 Mar 3;404(1):37-40
references.pmid: 9074633
references.citation: J Biol Chem. 2001 Aug 24;276(34):31772-9
references.pmid: 11429406
references.citation: Mol Cell Biol. 2000 Aug;20(16):5917-29
references.pmid: 10913175
references.citation: J Biol Chem. 1994 Apr 8;269(14):10524-8
references.pmid: 8144638
references.citation: Science. 1995 Sep 15;269(5230):1567-9
references.pmid: 7545311
references.citation: J Biol Chem. 2002 Nov 29;277(48):46687-95
references.pmid: 12351660
references.citation: J Biol Chem. 2000 Feb 4;275(5):3391-6
references.pmid: 10652331
references.citation: EMBO J. 2002 Jun 17;21(12):2977-89
references.pmid: 12065411
references.citation: Am J Physiol Cell Physiol. 2008 Apr;294(4):C931-44
references.pmid: 18216165
references.citation: Cell. 1988 Mar 25;52(6):801-10
references.pmid: 2450676
references.citation: Cell. 1987 Apr 10;49(1):1-4
references.pmid: 3030562
references.citation: Biol Direct. 2007 Jan 12;2:1
references.pmid: 17222345
references.citation: EMBO J. 1990 Oct;9(10):3241-52
references.pmid: 2170109
references.citation: Nat Rev Mol Cell Biol. 2006 Nov;7(11):833-46
references.pmid: 17057753
references.citation: J Biol Chem. 2002 May 17;277(20):17406-14
references.pmid: 11867627
references.citation: EMBO J. 1993 Oct;12(10):3789-98
references.pmid: 7691597
references.citation: Cell. 2004 Jun 11;117(6):699-711
references.pmid: 15186772
references.citation: J Cell Biol. 2003 Apr 14;161(1):143-53
references.pmid: 12682088
references.citation: Trends Biochem Sci. 1995 Nov;20(11):470-5
references.pmid: 8578591
references.citation: Science. 1996 Mar 22;271(5256):1718-23
references.pmid: 8596931
references.citation: Curr Opin Cell Biol. 2006 Dec;18(6):623-31
references.pmid: 17030123
references.citation: Biochem Biophys Res Commun. 2003 Nov 14;311(2):361-4
references.pmid: 14592422
references.citation: EMBO J. 1999 Jan 4;18(1):109-18
references.pmid: 9878055
references.citation: Curr Biol. 1999 May 20;9(10):505-11
references.pmid: 10339427
references.citation: J Biol Chem. 1998 Nov 27;273(48):31890-900
references.pmid: 9822658
references.citation: Biochem Biophys Res Commun. 2005 May 27;331(1):1-14
references.pmid: 15845350
references.citation: Nature. 1992 Sep 24;359(6393):336-9
references.pmid: 1383828
references.citation: Cell Growth Differ. 1995 Mar;6(3):303-7
references.pmid: 7794797
references.citation: Biochem Biophys Res Commun. 1998 Jan 6;242(1):32-8
references.pmid: 9439605
references.citation: J Cell Biol. 2008 Sep 22;182(6):1185-200
references.pmid: 18809727
references.citation: Biochem J. 2001 Mar 15;354(Pt 3):581-90
references.pmid: 11237862
references.citation: J Cell Biol. 1973 Dec;59(3):549-58
references.pmid: 4361656
references.citation: J Biol Chem. 1996 Nov 8;271(45):28086-96
references.pmid: 8910422
references.citation: J Cell Biol. 2005 Jan 3;168(1):127-39
references.pmid: 15623578
references.citation: Nature. 1999 Oct 7;401(6753):606-10
references.pmid: 10524630
references.citation: J Biol Chem. 1998 Apr 10;273(15):8691-8
references.pmid: 9535845
references.citation: Biochemistry. 2003 Jul 8;42(26):7904-14
references.pmid: 12834342
references.citation: J Cell Biol. 2003 Jan 6;160(1):137-46
references.pmid: 12515828
references.citation: J Cell Biol. 1999 Jan 25;144(2):315-24
references.pmid: 9922457
references.citation: J Biol Chem. 1995 May 5;270(18):10587-94
references.pmid: 7537734
references.citation: Nature. 1996 Aug 8;382(6591):555-9
references.pmid: 8700232
references.citation: Biochemistry. 2007 Jan 23;46(3):709-19
references.pmid: 17223692
references.citation: Cell. 1989 Jun 2;57(5):763-74
references.pmid: 2470512
references.citation: J Biol Chem. 2003 Apr 18;278(16):13968-74
references.pmid: 12582170
references.citation: J Biol Chem. 2003 Sep 5;278(36):34073-8
references.pmid: 12826681
references.citation: J Biol Chem. 2006 Apr 28;281(17):11972-80
references.pmid: 16507567
references.citation: EMBO J. 1996 Jun 17;15(12):3016-27
references.pmid: 8670803
references.citation: EMBO J. 2001 Nov 1;20(21):6037-49
references.pmid: 11689444
references.citation: EMBO J. 1994 Jul 1;13(13):3020-32
references.pmid: 7518772
references.citation: EMBO J. 2003 Aug 15;22(16):4121-31
references.pmid: 12912911
references.citation: Genes Cells. 2007 Jan;12(1):63-73
references.pmid: 17212655
references.citation: Nature. 1991 Jan 10;349(6305):172-5
references.pmid: 1702522
references.citation: Curr Biol. 1999 May 20;9(10):535-8
references.pmid: 10339428
references.citation: Eur J Biochem. 1997 May 1;245(3):693-700
references.pmid: 9183007
references.citation: BMC Cell Biol. 2001;2:8
references.pmid: 11401727