Serine dephosphorylation of receptor protein tyrosine phosphatase alpha in mitosis induces Src binding and activation.
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.
Vacaru
Andrei M
AM
Hubrecht Institute-KNAW and University Medical Center, Utrecht, Netherlands.
den Hertog
Jeroen
J
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*
D045163:Antibodies, Phospho-Specific; D010768:Phosphoserine; D019061:src-Family Kinases; D054648:Protein Phosphatase 2; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4
EMBO J. 2000 Mar 1;19(5):964-78
10698938
J Biol Chem. 1995 Sep 29;270(39):23126-31
7559456
EMBO J. 2002 Feb 15;21(4):493-503
11847098
Biochem J. 1997 Nov 1;327 ( Pt 3):867-76
9581568
J Biol Chem. 2002 Jun 14;277(24):21922-9
11923305
Mol Pharmacol. 2005 Apr;67(4):1206-13
15630078
FEBS Lett. 1997 Mar 3;404(1):37-40
9074633
J Biol Chem. 2001 Aug 24;276(34):31772-9
11429406
Mol Cell Biol. 2000 Aug;20(16):5917-29
10913175
J Biol Chem. 1994 Apr 8;269(14):10524-8
8144638
Science. 1995 Sep 15;269(5230):1567-9
7545311
J Biol Chem. 2002 Nov 29;277(48):46687-95
12351660
J Biol Chem. 2000 Feb 4;275(5):3391-6
10652331
EMBO J. 2002 Jun 17;21(12):2977-89
12065411
Am J Physiol Cell Physiol. 2008 Apr;294(4):C931-44
18216165
Cell. 1988 Mar 25;52(6):801-10
2450676
Cell. 1987 Apr 10;49(1):1-4
3030562
Biol Direct. 2007 Jan 12;2:1
17222345
EMBO J. 1990 Oct;9(10):3241-52
2170109
Nat Rev Mol Cell Biol. 2006 Nov;7(11):833-46
17057753
J Biol Chem. 2002 May 17;277(20):17406-14
11867627
EMBO J. 1993 Oct;12(10):3789-98
7691597
Cell. 2004 Jun 11;117(6):699-711
15186772
J Cell Biol. 2003 Apr 14;161(1):143-53
12682088
Trends Biochem Sci. 1995 Nov;20(11):470-5
8578591
Science. 1996 Mar 22;271(5256):1718-23
8596931
Curr Opin Cell Biol. 2006 Dec;18(6):623-31
17030123
Biochem Biophys Res Commun. 2003 Nov 14;311(2):361-4
14592422
EMBO J. 1999 Jan 4;18(1):109-18
9878055
Curr Biol. 1999 May 20;9(10):505-11
10339427
J Biol Chem. 1998 Nov 27;273(48):31890-900
9822658
Biochem Biophys Res Commun. 2005 May 27;331(1):1-14
15845350
Nature. 1992 Sep 24;359(6393):336-9
1383828
Cell Growth Differ. 1995 Mar;6(3):303-7
7794797
Biochem Biophys Res Commun. 1998 Jan 6;242(1):32-8
9439605
J Cell Biol. 2008 Sep 22;182(6):1185-200
18809727
Biochem J. 2001 Mar 15;354(Pt 3):581-90
11237862
J Cell Biol. 1973 Dec;59(3):549-58
4361656
J Biol Chem. 1996 Nov 8;271(45):28086-96
8910422
J Cell Biol. 2005 Jan 3;168(1):127-39
15623578
Nature. 1999 Oct 7;401(6753):606-10
10524630
J Biol Chem. 1998 Apr 10;273(15):8691-8
9535845
Biochemistry. 2003 Jul 8;42(26):7904-14
12834342
J Cell Biol. 2003 Jan 6;160(1):137-46
12515828
J Cell Biol. 1999 Jan 25;144(2):315-24
9922457
J Biol Chem. 1995 May 5;270(18):10587-94
7537734
Nature. 1996 Aug 8;382(6591):555-9
8700232
Biochemistry. 2007 Jan 23;46(3):709-19
17223692
Cell. 1989 Jun 2;57(5):763-74
2470512
J Biol Chem. 2003 Apr 18;278(16):13968-74
12582170
J Biol Chem. 2003 Sep 5;278(36):34073-8
12826681
J Biol Chem. 2006 Apr 28;281(17):11972-80
16507567
EMBO J. 1996 Jun 17;15(12):3016-27
8670803
EMBO J. 2001 Nov 1;20(21):6037-49
11689444
EMBO J. 1994 Jul 1;13(13):3020-32
7518772
EMBO J. 2003 Aug 15;22(16):4121-31
12912911
Genes Cells. 2007 Jan;12(1):63-73
17212655
Nature. 1991 Jan 10;349(6305):172-5
1702522
Curr Biol. 1999 May 20;9(10):535-8
10339428
Eur J Biochem. 1997 May 1;245(3):693-700
9183007
BMC Cell Biol. 2001;2:8
11401727