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. 1992 Dec;12(12):5824–5833. doi: 10.1128/mcb.12.12.5824

The SH2 and SH3 domain-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors.

W Li 1, P Hu 1, E Y Skolnik 1, A Ullrich 1, J Schlessinger 1
PMCID: PMC360522  PMID: 1333047

Abstract

Signalling proteins such as phospholipase C-gamma (PLC-gamma) or GTPase-activating protein (GAP) of ras contain conserved regions of approximately 100 amino acids termed src homology 2 (SH2) domains. SH2 domains were shown to be responsible for mediating association between signalling proteins and tyrosine-phosphorylated proteins, including growth factor receptors. Nck is an ubiquitously expressed protein consisting exclusively of one SH2 and three SH3 domains. Here we show that epidermal growth factor or platelet-derived growth factor stimulation of intact human or murine cells leads to phosphorylation of Nck protein on tyrosine, serine, and threonine residues. Similar stimulation of Nck phosphorylation was detected upon activation of rat basophilic leukemia RBL-2H3 cells by cross-linking of the high-affinity immunoglobulin E receptors (Fc epsilon RI). Ligand-activated, tyrosine-autophosphorylated platelet-derived growth factor or epidermal growth factor receptors were coimmunoprecipitated with anti-Nck antibodies, and the association with either receptor molecule was mediated by the SH2 domain of Nck. Addition of phorbol ester was also able to stimulate Nck phosphorylation on serine residues. However, growth factor-induced serine/threonine phosphorylation of Nck was not mediated by protein kinase C. Interestingly, approximately fivefold overexpression of Nck in NIH 3T3 cells resulted in formation of oncogenic foci. These results show that Nck is an oncogenic protein and a common target for the action of different surface receptors. Nck probably functions as an adaptor protein which links surface receptors with tyrosine kinase activity to downstream signalling pathways involved in the control of cell proliferation.

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Selected References

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

  1. Aaronson S. A. Growth factors and cancer. Science. 1991 Nov 22;254(5035):1146–1153. doi: 10.1126/science.1659742. [DOI] [PubMed] [Google Scholar]
  2. Bond V. C., Wold B. Poly-L-ornithine-mediated transformation of mammalian cells. Mol Cell Biol. 1987 Jun;7(6):2286–2293. doi: 10.1128/mcb.7.6.2286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Bustelo X. R., Ledbetter J. A., Barbacid M. Product of vav proto-oncogene defines a new class of tyrosine protein kinase substrates. Nature. 1992 Mar 5;356(6364):68–71. doi: 10.1038/356068a0. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Chou M. M., Fajardo J. E., Hanafusa H. The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels. Mol Cell Biol. 1992 Dec;12(12):5834–5842. doi: 10.1128/mcb.12.12.5834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  9. Davis S., Lu M. L., Lo S. H., Lin S., Butler J. A., Druker B. J., Roberts T. M., An Q., Chen L. B. Presence of an SH2 domain in the actin-binding protein tensin. Science. 1991 May 3;252(5006):712–715. doi: 10.1126/science.1708917. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Gould K. L., Hunter T. Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity. Mol Cell Biol. 1988 Aug;8(8):3345–3356. doi: 10.1128/mcb.8.8.3345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heldin C. H. SH2 domains: elements that control protein interactions during signal transduction. Trends Biochem Sci. 1991 Dec;16(12):450–452. doi: 10.1016/0968-0004(91)90175-u. [DOI] [PubMed] [Google Scholar]
  13. Hu P., Margolis B., Skolnik E. Y., Lammers R., Ullrich A., Schlessinger J. Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors. Mol Cell Biol. 1992 Mar;12(3):981–990. doi: 10.1128/mcb.12.3.981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Katzav S., Cleveland J. L., Heslop H. E., Pulido D. Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential. Mol Cell Biol. 1991 Apr;11(4):1912–1920. doi: 10.1128/mcb.11.4.1912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Katzav S., Martin-Zanca D., Barbacid M. vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells. EMBO J. 1989 Aug;8(8):2283–2290. doi: 10.1002/j.1460-2075.1989.tb08354.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kazlauskas A., Cooper J. A. Phosphorylation of the PDGF receptor beta subunit creates a tight binding site for phosphatidylinositol 3 kinase. EMBO J. 1990 Oct;9(10):3279–3286. doi: 10.1002/j.1460-2075.1990.tb07527.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kazlauskas A., Kashishian A., Cooper J. A., Valius M. GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit. Mol Cell Biol. 1992 Jun;12(6):2534–2544. doi: 10.1128/mcb.12.6.2534. [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. Kypta R. M., Goldberg Y., Ulug E. T., Courtneidge S. A. Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell. 1990 Aug 10;62(3):481–492. doi: 10.1016/0092-8674(90)90013-5. [DOI] [PubMed] [Google Scholar]
  21. Lehmann J. M., Riethmüller G., Johnson J. P. Nck, a melanoma cDNA encoding a cytoplasmic protein consisting of the src homology units SH2 and SH3. Nucleic Acids Res. 1990 Feb 25;18(4):1048–1048. doi: 10.1093/nar/18.4.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li W., Deanin G. G., Margolis B., Schlessinger J., Oliver J. M. Fc epsilon R1-mediated tyrosine phosphorylation of multiple proteins, including phospholipase C gamma 1 and the receptor beta gamma 2 complex, in RBL-2H3 rat basophilic leukemia cells. Mol Cell Biol. 1992 Jul;12(7):3176–3182. doi: 10.1128/mcb.12.7.3176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li W., Yeung Y. G., Stanley E. R. Tyrosine phosphorylation of a common 57-kDa protein in growth factor-stimulated and -transformed cells. J Biol Chem. 1991 Apr 15;266(11):6808–6814. [PubMed] [Google Scholar]
  24. Liu F. T., Bohn J. W., Ferry E. L., Yamamoto H., Molinaro C. A., Sherman L. A., Klinman N. R., Katz D. H. Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization. J Immunol. 1980 Jun;124(6):2728–2737. [PubMed] [Google Scholar]
  25. 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]
  26. Margolis B., Hu P., Katzav S., Li W., Oliver J. M., Ullrich A., Weiss A., Schlessinger J. Tyrosine phosphorylation of vav proto-oncogene product containing SH2 domain and transcription factor motifs. Nature. 1992 Mar 5;356(6364):71–74. doi: 10.1038/356071a0. [DOI] [PubMed] [Google Scholar]
  27. Margolis B. Proteins with SH2 domains: transducers in the tyrosine kinase signaling pathway. Cell Growth Differ. 1992 Jan;3(1):73–80. [PubMed] [Google Scholar]
  28. Mayer B. J., Hamaguchi M., Hanafusa H. A novel viral oncogene with structural similarity to phospholipase C. Nature. 1988 Mar 17;332(6161):272–275. doi: 10.1038/332272a0. [DOI] [PubMed] [Google Scholar]
  29. Mayer B. J., Hanafusa H. Association of the v-crk oncogene product with phosphotyrosine-containing proteins and protein kinase activity. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2638–2642. doi: 10.1073/pnas.87.7.2638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Meisenhelder J., Hunter T. The SH2/SH3 domain-containing protein Nck is recognized by certain anti-phospholipase C-gamma 1 monoclonal antibodies, and its phosphorylation on tyrosine is stimulated by platelet-derived growth factor and epidermal growth factor treatment. Mol Cell Biol. 1992 Dec;12(12):5843–5856. doi: 10.1128/mcb.12.12.5843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Park D. J., Min H. K., Rhee S. G. IgE-induced tyrosine phosphorylation of phospholipase C-gamma 1 in rat basophilic leukemia cells. J Biol Chem. 1991 Dec 25;266(36):24237–24240. [PubMed] [Google Scholar]
  35. Park D., Rhee S. G. Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP. Mol Cell Biol. 1992 Dec;12(12):5816–5823. doi: 10.1128/mcb.12.12.5816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Schlessinger J. Signal transduction by allosteric receptor oligomerization. Trends Biochem Sci. 1988 Nov;13(11):443–447. doi: 10.1016/0968-0004(88)90219-8. [DOI] [PubMed] [Google Scholar]
  39. Shen S. H., Bastien L., Posner B. I., Chrétien P. A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases. Nature. 1991 Aug 22;352(6337):736–739. doi: 10.1038/352736a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Suh P. G., Ryu S. H., Moon K. H., Suh H. W., Rhee S. G. Inositol phospholipid-specific phospholipase C: complete cDNA and protein sequences and sequence homology to tyrosine kinase-related oncogene products. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5419–5423. doi: 10.1073/pnas.85.15.5419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Thomas G. MAP kinase by any other name smells just as sweet. Cell. 1992 Jan 10;68(1):3–6. doi: 10.1016/0092-8674(92)90199-m. [DOI] [PubMed] [Google Scholar]
  44. Trahey M., Wong G., Halenbeck R., Rubinfeld B., Martin G. A., Ladner M., Long C. M., Crosier W. J., Watt K., Koths K. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. doi: 10.1126/science.3201259. [DOI] [PubMed] [Google Scholar]
  45. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  46. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  47. Yi T. L., Cleveland J. L., Ihle J. N. Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13. Mol Cell Biol. 1992 Feb;12(2):836–846. doi: 10.1128/mcb.12.2.836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. van der Geer P., Hunter T. Tyrosine 706 and 807 phosphorylation site mutants in the murine colony-stimulating factor-1 receptor are unaffected in their ability to bind or phosphorylate phosphatidylinositol-3 kinase but show differential defects in their ability to induce early response gene transcription. Mol Cell Biol. 1991 Sep;11(9):4698–4709. doi: 10.1128/mcb.11.9.4698. [DOI] [PMC free article] [PubMed] [Google Scholar]

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