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. 1993 Aug;13(8):4600–4608. doi: 10.1128/mcb.13.8.4600

A novel recognition motif for phosphatidylinositol 3-kinase binding mediates its association with the hepatocyte growth factor/scatter factor receptor.

C Ponzetto 1, A Bardelli 1, F Maina 1, P Longati 1, G Panayotou 1, R Dhand 1, M D Waterfield 1, P M Comoglio 1
PMCID: PMC360084  PMID: 7687741

Abstract

The pleiotropic effects (mitogenesis, motogenesis, and morphogenesis) elicited by hepatocyte growth factor/scatter factor (HGF/SF) are mediated by the activation of the tyrosine kinase receptor encoded by the MET proto-oncogene. Following autophosphorylation, the receptor associates with the p85/110 phosphatidylinositol (PI) 3-kinase complex in vivo and in vitro. By a combination of two complementary approaches, competition with synthetic phosphopeptides and association with Tyr-Phe receptor mutants, we have identified Y-1349 and Y-1356 in the HGF/SF receptor as the binding sites for PI 3-kinase. Y-1349VHV and Y-1356VNV do not conform to the canonical consensus sequence YXXM for PI 3-kinase binding and thus define YVXV as a novel recognition motif. Y-1349 and Y-1356 are located within the C-terminal portion of the HGF/SF receptor and are phosphorylation sites. The affinity of the N- and C-terminal src homology region 2 (SH2) domains of p85 for the phosphopeptides including Y-1349 and Y-1356 is 2 orders of magnitude lower than that measured for Y-751 in the platelet-derived growth factor receptor binding site. However, the closely spaced duplication of the novel recognition motif in the native HGF/SF receptor may allow binding with both SH2 domains of p85, thus generating an efficient docking site for PI 3-kinase. In agreement with this model, we have observed that a phosphopeptide including both Y-1349 and Y-1356 activates PI 3-kinase in vitro.

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Selected References

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

  1. Backer J. M., Myers M. G., Jr, Shoelson S. E., Chin D. J., Sun X. J., Miralpeix M., Hu P., Margolis B., Skolnik E. Y., Schlessinger J. Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J. 1992 Sep;11(9):3469–3479. doi: 10.1002/j.1460-2075.1992.tb05426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bardelli A., Maina F., Gout I., Fry M. J., Waterfield M. D., Comoglio P. M., Ponzetto C. Autophosphorylation promotes complex formation of recombinant hepatocyte growth factor receptor with cytoplasmic effectors containing SH2 domains. Oncogene. 1992 Oct;7(10):1973–1978. [PubMed] [Google Scholar]
  3. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  4. Carpenter C. L., Auger K. R., Chanudhuri M., Yoakim M., Schaffhausen B., Shoelson S., Cantley L. C. Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit. J Biol Chem. 1993 May 5;268(13):9478–9483. [PubMed] [Google Scholar]
  5. Carpenter C. L., Cantley L. C. Phosphoinositide kinases. Biochemistry. 1990 Dec 25;29(51):11147–11156. doi: 10.1021/bi00503a001. [DOI] [PubMed] [Google Scholar]
  6. Comoglio P. M. Structure, biosynthesis and biochemical properties of the HGF receptor in normal and malignant cells. EXS. 1993;65:131–165. [PubMed] [Google Scholar]
  7. Escobedo J. A., Kaplan D. R., Kavanaugh W. M., Turck C. W., Williams L. T. A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine. Mol Cell Biol. 1991 Feb;11(2):1125–1132. doi: 10.1128/mcb.11.2.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Escobedo J. A., Navankasattusas S., Kavanaugh W. M., Milfay D., Fried V. A., Williams L. T. cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor. Cell. 1991 Apr 5;65(1):75–82. doi: 10.1016/0092-8674(91)90409-r. [DOI] [PubMed] [Google Scholar]
  9. Ferracini R., Longati P., Naldini L., Vigna E., Comoglio P. M. Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase. J Biol Chem. 1991 Oct 15;266(29):19558–19564. [PubMed] [Google Scholar]
  10. Graziani A., Gramaglia D., Cantley L. C., Comoglio P. M. The tyrosine-phosphorylated hepatocyte growth factor/scatter factor receptor associates with phosphatidylinositol 3-kinase. J Biol Chem. 1991 Nov 25;266(33):22087–22090. [PubMed] [Google Scholar]
  11. Graziani A., Gramaglia D., dalla Zonca P., Comoglio P. M. Hepatocyte growth factor/scatter factor stimulates the Ras-guanine nucleotide exchanger. J Biol Chem. 1993 May 5;268(13):9165–9168. [PubMed] [Google Scholar]
  12. 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]
  13. Kan M., Zhang G. H., Zarnegar R., Michalopoulos G., Myoken Y., McKeehan W. L., Stevens J. I. Hepatocyte growth factor/hepatopoietin A stimulates the growth of rat kidney proximal tubule epithelial cells (RPTE), rat nonparenchymal liver cells, human melanoma cells, mouse keratinocytes and stimulates anchorage-independent growth of SV-40 transformed RPTE. Biochem Biophys Res Commun. 1991 Jan 15;174(1):331–337. doi: 10.1016/0006-291x(91)90524-b. [DOI] [PubMed] [Google Scholar]
  14. Karlsson R., Michaelsson A., Mattsson L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J Immunol Methods. 1991 Dec 15;145(1-2):229–240. doi: 10.1016/0022-1759(91)90331-9. [DOI] [PubMed] [Google Scholar]
  15. Kashishian A., Kazlauskas A., Cooper J. A. Phosphorylation sites in the PDGF receptor with different specificities for binding GAP and PI3 kinase in vivo. EMBO J. 1992 Apr;11(4):1373–1382. doi: 10.1002/j.1460-2075.1992.tb05182.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kavanaugh W. M., Klippel A., Escobedo J. A., Williams L. T. Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells. Mol Cell Biol. 1992 Aug;12(8):3415–3424. doi: 10.1128/mcb.12.8.3415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kazlauskas A., Cooper J. A. Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins. Cell. 1989 Sep 22;58(6):1121–1133. doi: 10.1016/0092-8674(89)90510-2. [DOI] [PubMed] [Google Scholar]
  18. Klippel A., Escobedo J. A., Fantl W. J., Williams L. T. The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor. Mol Cell Biol. 1992 Apr;12(4):1451–1459. doi: 10.1128/mcb.12.4.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  20. Mohammadi M., Honegger A. M., Rotin D., Fischer R., Bellot F., Li W., Dionne C. A., Jaye M., Rubinstein M., Schlessinger J. A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1. Mol Cell Biol. 1991 Oct;11(10):5068–5078. doi: 10.1128/mcb.11.10.5068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Naldini L., Weidner K. M., Vigna E., Gaudino G., Bardelli A., Ponzetto C., Narsimhan R. P., Hartmann G., Zarnegar R., Michalopoulos G. K. Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO J. 1991 Oct;10(10):2867–2878. doi: 10.1002/j.1460-2075.1991.tb07836.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Otsu M., Hiles I., Gout I., Fry M. J., Ruiz-Larrea F., Panayotou G., Thompson A., Dhand R., Hsuan J., Totty N. Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. Cell. 1991 Apr 5;65(1):91–104. doi: 10.1016/0092-8674(91)90411-q. [DOI] [PubMed] [Google Scholar]
  23. Panayotou G., Bax B., Gout I., Federwisch M., Wroblowski B., Dhand R., Fry M. J., Blundell T. L., Wollmer A., Waterfield M. D. Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes. EMBO J. 1992 Dec;11(12):4261–4272. doi: 10.1002/j.1460-2075.1992.tb05524.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Panayotou G., Gish G., End P., Truong O., Gout I., Dhand R., Fry M. J., Hiles I., Pawson T., Waterfield M. D. Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach. Mol Cell Biol. 1993 Jun;13(6):3567–3576. doi: 10.1128/mcb.13.6.3567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Panayotou G., Waterfield M. D. Phosphatidyl-inositol 3-kinase: a key enzyme in diverse signalling processes. Trends Cell Biol. 1992 Dec;2(12):358–360. doi: 10.1016/0962-8924(92)90042-l. [DOI] [PubMed] [Google Scholar]
  26. Ponzetto C., Giordano S., Peverali F., Della Valle G., Abate M. L., Vaula G., Comoglio P. M. c-met is amplified but not mutated in a cell line with an activated met tyrosine kinase. Oncogene. 1991 Apr;6(4):553–559. [PubMed] [Google Scholar]
  27. Reedijk M., Liu X. Q., Pawson T. Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor. Mol Cell Biol. 1990 Nov;10(11):5601–5608. doi: 10.1128/mcb.10.11.5601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Reedijk M., Liu X., van der Geer P., Letwin K., Waterfield M. D., Hunter T., Pawson T. Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions. EMBO J. 1992 Apr;11(4):1365–1372. doi: 10.1002/j.1460-2075.1992.tb05181.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rotin D., Margolis B., Mohammadi M., Daly R. J., Daum G., Li N., Fischer E. H., Burgess W. H., Ullrich A., Schlessinger J. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma. EMBO J. 1992 Feb;11(2):559–567. doi: 10.1002/j.1460-2075.1992.tb05087.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rubin J. S., Chan A. M., Bottaro D. P., Burgess W. H., Taylor W. G., Cech A. C., Hirschfield D. W., Wong J., Miki T., Finch P. W. A broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth factor. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):415–419. doi: 10.1073/pnas.88.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Skolnik E. Y., Margolis B., Mohammadi M., Lowenstein E., Fischer R., Drepps A., Ullrich A., Schlessinger J. Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases. Cell. 1991 Apr 5;65(1):83–90. doi: 10.1016/0092-8674(91)90410-z. [DOI] [PubMed] [Google Scholar]
  32. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  33. Sognier M. A., Neft R. E., Roe A. L., Eberle R. L., Belli J. A. Dot-blot hybridization: quantitative analysis with direct beta counting. Biotechniques. 1991 Oct;11(4):520–525. [PubMed] [Google Scholar]
  34. Stoker M., Gherardi E., Perryman M., Gray J. Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature. 1987 May 21;327(6119):239–242. doi: 10.1038/327239a0. [DOI] [PubMed] [Google Scholar]
  35. Turck C. W. Identification of phosphotyrosine residues in peptides by high performance liquid chromatography on-line derivative spectroscopy. Pept Res. 1992 May-Jun;5(3):156–160. [PubMed] [Google Scholar]
  36. Weidner K. M., Behrens J., Vandekerckhove J., Birchmeier W. Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J Cell Biol. 1990 Nov;111(5 Pt 1):2097–2108. doi: 10.1083/jcb.111.5.2097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Whitman M., Kaplan D. R., Schaffhausen B., Cantley L., Roberts T. M. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature. 1985 May 16;315(6016):239–242. doi: 10.1038/315239a0. [DOI] [PubMed] [Google Scholar]

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