Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1994 Jul 1;13(13):3020–3032. doi: 10.1002/j.1460-2075.1994.tb06601.x

Phosphorylation of receptor protein-tyrosine phosphatase alpha on Tyr789, a binding site for the SH3-SH2-SH3 adaptor protein GRB-2 in vivo.

J den Hertog 1, S Tracy 1, T Hunter 1
PMCID: PMC395191  PMID: 7518772

Abstract

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) is a transmembrane protein with a short extracellular domain (123 amino acids) and two cytoplasmically localized protein-tyrosine phosphatase (PTP) domains. Here we report that RPTP alpha is constitutively phosphorylated on tyrosine in NIH 3T3 mouse fibroblasts. The in vivo tyrosine phosphorylation site was localized to the C-terminus of RPTP alpha by phosphopeptide mapping experiments using in vivo and in vitro 32P-labeled RPTP alpha. The identity of this site as Tyr789, located five residues from the C-terminus, was confirmed by site-directed mutagenesis. Transient overexpression of c-Src together with RPTP alpha in human embryonic kidney 293 cells increased phosphorylation of Tyr789, suggesting that c-Src may phosphorylate RPTP alpha in vivo. RPTP alpha had autodephosphorylation activity in vitro. When expressed in 293 cells the level of Tyr789 phosphorylation was higher in a non-functional mutant of RPTP alpha than in wild type RPTP alpha, indicating that RPTP alpha may have autodephosphorylation activity in vivo as well. The sequence on the C-terminal side of Tyr789 (YANF) fits the consensus binding site for the SH3-SH2-SH3 adaptor protein GRB2 (YXNX). We show that RPTP alpha, but not a mutant of RPTP alpha with a Tyr-->Phe mutation at position 789, bound to GRB2 in vitro. In addition, RPTP alpha co-immunoprecipitated with GRB2 from NIH 3T3 cells, demonstrating that GRB2 bound to RPTP alpha in vivo. The guanine nucleotide releasing factor for the Ras GTPase, Son of sevenless (Sos), which associates with GRB2 via its SH3 domains, was not detected in RPTP alpha immunoprecipitates. Our results suggest a role for RPTP alpha in attenuation of GRB2-mediated signaling.

Full text

PDF
3020

Images in this article

3021Image
on p.3021
3022Image
on p.3022
3023Image
on p.3023
3023Image
on p.3023
3024Image
on p.3024
3024Image
on p.3024
3025Image
on p.3025
3026Image
on p.3026
3026Image
on p.3026
3027Image
on p.3027
3028Image
on p.3028

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Autero M., Saharinen J., Pessa-Morikawa T., Soula-Rothhut M., Oetken C., Gassmann M., Bergman M., Alitalo K., Burn P., Gahmberg C. G. Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase. Mol Cell Biol. 1994 Feb;14(2):1308–1321. doi: 10.1128/mcb.14.2.1308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  3. Brady-Kalnay S. M., Flint A. J., Tonks N. K. Homophilic binding of PTP mu, a receptor-type protein tyrosine phosphatase, can mediate cell-cell aggregation. J Cell Biol. 1993 Aug;122(4):961–972. doi: 10.1083/jcb.122.4.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brautigan D. L., Pinault F. M. Activation of membrane protein-tyrosine phosphatase involving cAMP- and Ca2+/phospholipid-dependent protein kinases. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6696–6700. doi: 10.1073/pnas.88.15.6696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Charbonneau H., Tonks N. K. 1002 protein phosphatases? Annu Rev Cell Biol. 1992;8:463–493. doi: 10.1146/annurev.cb.08.110192.002335. [DOI] [PubMed] [Google Scholar]
  6. Chardin P., Camonis J. H., Gale N. W., van Aelst L., Schlessinger J., Wigler M. H., Bar-Sagi D. Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science. 1993 May 28;260(5112):1338–1343. doi: 10.1126/science.8493579. [DOI] [PubMed] [Google Scholar]
  7. Cicchetti P., Mayer B. J., Thiel G., Baltimore D. Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho. Science. 1992 Aug 7;257(5071):803–806. doi: 10.1126/science.1379745. [DOI] [PubMed] [Google Scholar]
  8. Clark S. G., Stern M. J., Horvitz H. R. C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature. 1992 Mar 26;356(6367):340–344. doi: 10.1038/356340a0. [DOI] [PubMed] [Google Scholar]
  9. Egan S. E., Giddings B. W., Brooks M. W., Buday L., Sizeland A. M., Weinberg R. A. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature. 1993 May 6;363(6424):45–51. doi: 10.1038/363045a0. [DOI] [PubMed] [Google Scholar]
  10. Feng G. S., Hui C. C., Pawson T. SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases. Science. 1993 Mar 12;259(5101):1607–1611. doi: 10.1126/science.8096088. [DOI] [PubMed] [Google Scholar]
  11. Fischer E. H., Charbonneau H., Tonks N. K. Protein tyrosine phosphatases: a diverse family of intracellular and transmembrane enzymes. Science. 1991 Jul 26;253(5018):401–406. doi: 10.1126/science.1650499. [DOI] [PubMed] [Google Scholar]
  12. Flint A. J., Gebbink M. F., Franza B. R., Jr, Hill D. E., Tonks N. K. Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation. EMBO J. 1993 May;12(5):1937–1946. doi: 10.1002/j.1460-2075.1993.tb05843.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gebbink M. F., Zondag G. C., Wubbolts R. W., Beijersbergen R. L., van Etten I., Moolenaar W. H. Cell-cell adhesion mediated by a receptor-like protein tyrosine phosphatase. J Biol Chem. 1993 Aug 5;268(22):16101–16104. [PubMed] [Google Scholar]
  14. Gebbink M. F., van Etten I., Hateboer G., Suijkerbuijk R., Beijersbergen R. L., Geurts van Kessel A., Moolenaar W. H. Cloning, expression and chromosomal localization of a new putative receptor-like protein tyrosine phosphatase. FEBS Lett. 1991 Sep 23;290(1-2):123–130. doi: 10.1016/0014-5793(91)81241-y. [DOI] [PubMed] [Google Scholar]
  15. Gout I., Dhand R., Hiles I. D., Fry M. J., Panayotou G., Das P., Truong O., Totty N. F., Hsuan J., Booker G. W. The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell. 1993 Oct 8;75(1):25–36. [PubMed] [Google Scholar]
  16. Green S., Issemann I., Sheer E. A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res. 1988 Jan 11;16(1):369–369. doi: 10.1093/nar/16.1.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  18. Guan K. L., Dixon J. E. Evidence for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate intermediate. J Biol Chem. 1991 Sep 15;266(26):17026–17030. [PubMed] [Google Scholar]
  19. Guan K. L., Dixon J. E. Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia. Science. 1990 Aug 3;249(4968):553–556. doi: 10.1126/science.2166336. [DOI] [PubMed] [Google Scholar]
  20. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  21. Jiang Y. P., Wang H., D'Eustachio P., Musacchio J. M., Schlessinger J., Sap J. Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region. Mol Cell Biol. 1993 May;13(5):2942–2951. doi: 10.1128/mcb.13.5.2942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kazlauskas A., Feng G. S., Pawson T., Valius M. The 64-kDa protein that associates with the platelet-derived growth factor receptor beta subunit via Tyr-1009 is the SH2-containing phosphotyrosine phosphatase Syp. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6939–6943. doi: 10.1073/pnas.90.15.6939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koff A., Giordano A., Desai D., Yamashita K., Harper J. W., Elledge S., Nishimoto T., Morgan D. O., Franza B. R., Roberts J. M. Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science. 1992 Sep 18;257(5077):1689–1694. doi: 10.1126/science.1388288. [DOI] [PubMed] [Google Scholar]
  24. Krueger N. X., Streuli M., Saito H. Structural diversity and evolution of human receptor-like protein tyrosine phosphatases. EMBO J. 1990 Oct;9(10):3241–3252. doi: 10.1002/j.1460-2075.1990.tb07523.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  26. Lechleider R. J., Freeman R. M., Jr, Neel B. G. Tyrosyl phosphorylation and growth factor receptor association of the human corkscrew homologue, SH-PTP2. J Biol Chem. 1993 Jun 25;268(18):13434–13438. [PubMed] [Google Scholar]
  27. Lechleider R. J., Sugimoto S., Bennett A. M., Kashishian A. S., Cooper J. A., Shoelson S. E., Walsh C. T., Neel B. G. Activation of the SH2-containing phosphotyrosine phosphatase SH-PTP2 by its binding site, phosphotyrosine 1009, on the human platelet-derived growth factor receptor. J Biol Chem. 1993 Oct 15;268(29):21478–21481. [PubMed] [Google Scholar]
  28. Li N., Batzer A., Daly R., Yajnik V., Skolnik E., Chardin P., Bar-Sagi D., Margolis B., Schlessinger J. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature. 1993 May 6;363(6424):85–88. doi: 10.1038/363085a0. [DOI] [PubMed] [Google Scholar]
  29. Li W., Nishimura R., Kashishian A., Batzer A. G., Kim W. J., Cooper J. A., Schlessinger J. A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase. Mol Cell Biol. 1994 Jan;14(1):509–517. doi: 10.1128/mcb.14.1.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu X., Brodeur S. R., Gish G., Songyang Z., Cantley L. C., Laudano A. P., Pawson T. Regulation of c-Src tyrosine kinase activity by the Src SH2 domain. Oncogene. 1993 May;8(5):1119–1126. [PubMed] [Google Scholar]
  32. Lorenz U., Ravichandran K. S., Pei D., Walsh C. T., Burakoff S. J., Neel B. G. Lck-dependent tyrosyl phosphorylation of the phosphotyrosine phosphatase SH-PTP1 in murine T cells. Mol Cell Biol. 1994 Mar;14(3):1824–1834. doi: 10.1128/mcb.14.3.1824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lowenstein E. J., Daly R. J., Batzer A. G., Li W., Margolis B., Lammers R., Ullrich A., Skolnik E. Y., Bar-Sagi D., Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. doi: 10.1016/0092-8674(92)90167-b. [DOI] [PubMed] [Google Scholar]
  34. Matthews R. J., Cahir E. D., Thomas M. L. Identification of an additional member of the protein-tyrosine-phosphatase family: evidence for alternative splicing in the tyrosine phosphatase domain. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4444–4448. doi: 10.1073/pnas.87.12.4444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Olivier J. P., Raabe T., Henkemeyer M., Dickson B., Mbamalu G., Margolis B., Schlessinger J., Hafen E., Pawson T. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos. Cell. 1993 Apr 9;73(1):179–191. doi: 10.1016/0092-8674(93)90170-u. [DOI] [PubMed] [Google Scholar]
  36. Ostergaard H. L., Trowbridge I. S. Negative regulation of CD45 protein tyrosine phosphatase activity by ionomycin in T cells. Science. 1991 Sep 20;253(5026):1423–1425. doi: 10.1126/science.1654595. [DOI] [PubMed] [Google Scholar]
  37. Pawson T., Schlessingert J. SH2 and SH3 domains. Curr Biol. 1993 Jul 1;3(7):434–442. doi: 10.1016/0960-9822(93)90350-w. [DOI] [PubMed] [Google Scholar]
  38. Pot D. A., Dixon J. E. Active site labeling of a receptor-like protein tyrosine phosphatase. J Biol Chem. 1992 Jan 5;267(1):140–143. [PubMed] [Google Scholar]
  39. Sap J., D'Eustachio P., Givol D., Schlessinger J. Cloning and expression of a widely expressed receptor tyrosine phosphatase. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6112–6116. doi: 10.1073/pnas.87.16.6112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sap J., Jiang Y. P., Friedlander D., Grumet M., Schlessinger J. Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding. Mol Cell Biol. 1994 Jan;14(1):1–9. doi: 10.1128/mcb.14.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schlessinger J., Ullrich A. Growth factor signaling by receptor tyrosine kinases. Neuron. 1992 Sep;9(3):383–391. doi: 10.1016/0896-6273(92)90177-f. [DOI] [PubMed] [Google Scholar]
  42. Simon M. A., Dodson G. S., Rubin G. M. An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell. 1993 Apr 9;73(1):169–177. doi: 10.1016/0092-8674(93)90169-q. [DOI] [PubMed] [Google Scholar]
  43. Skolnik E. Y., Batzer A., Li N., Lee C. H., Lowenstein E., Mohammadi M., Margolis B., Schlessinger J. The function of GRB2 in linking the insulin receptor to Ras signaling pathways. Science. 1993 Jun 25;260(5116):1953–1955. doi: 10.1126/science.8316835. [DOI] [PubMed] [Google Scholar]
  44. Songyang Z., Shoelson S. E., Chaudhuri M., Gish G., Pawson T., Haser W. G., King F., Roberts T., Ratnofsky S., Lechleider R. J. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. doi: 10.1016/0092-8674(93)90404-e. [DOI] [PubMed] [Google Scholar]
  45. Stover D. R., Charbonneau H., Tonks N. K., Walsh K. A. Protein-tyrosine-phosphatase CD45 is phosphorylated transiently on tyrosine upon activation of Jurkat T cells. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7704–7707. doi: 10.1073/pnas.88.17.7704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Streuli M., Krueger N. X., Thai T., Tang M., Saito H. Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR. EMBO J. 1990 Aug;9(8):2399–2407. doi: 10.1002/j.1460-2075.1990.tb07415.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Streuli M., Krueger N. X., Tsai A. Y., Saito H. A family of receptor-linked protein tyrosine phosphatases in humans and Drosophila. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8698–8702. doi: 10.1073/pnas.86.22.8698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tan X., Stover D. R., Walsh K. A. Demonstration of protein tyrosine phosphatase activity in the second of two homologous domains of CD45. J Biol Chem. 1993 Apr 5;268(10):6835–6838. [PubMed] [Google Scholar]
  49. Vogel W., Lammers R., Huang J., Ullrich A. Activation of a phosphotyrosine phosphatase by tyrosine phosphorylation. Science. 1993 Mar 12;259(5101):1611–1614. doi: 10.1126/science.7681217. [DOI] [PubMed] [Google Scholar]
  50. Wang Y., Pallen C. J. The receptor-like protein tyrosine phosphatase HPTP alpha has two active catalytic domains with distinct substrate specificities. EMBO J. 1991 Nov;10(11):3231–3237. doi: 10.1002/j.1460-2075.1991.tb04886.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yamada A., Streuli M., Saito H., Rothstein D. M., Schlossman S. F., Morimoto C. Effect of activation of protein kinase C on CD45 isoform expression and CD45 protein tyrosine phosphatase activity in T cells. Eur J Immunol. 1990 Aug;20(8):1655–1660. doi: 10.1002/eji.1830200806. [DOI] [PubMed] [Google Scholar]
  52. Yi T., Ihle J. N. Association of hematopoietic cell phosphatase with c-Kit after stimulation with c-Kit ligand. Mol Cell Biol. 1993 Jun;13(6):3350–3358. doi: 10.1128/mcb.13.6.3350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zheng X. M., Wang Y., Pallen C. J. Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase. Nature. 1992 Sep 24;359(6393):336–339. doi: 10.1038/359336a0. [DOI] [PubMed] [Google Scholar]
  54. den Hertog J., Pals C. E., Peppelenbosch M. P., Tertoolen L. G., de Laat S. W., Kruijer W. Receptor protein tyrosine phosphatase alpha activates pp60c-src and is involved in neuronal differentiation. EMBO J. 1993 Oct;12(10):3789–3798. doi: 10.1002/j.1460-2075.1993.tb06057.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. van der Geer P., Hunter T. Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts. EMBO J. 1993 Dec 15;12(13):5161–5172. doi: 10.1002/j.1460-2075.1993.tb06211.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES