Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1996 Jan 1;183(1):179–186. doi: 10.1084/jem.183.1.179

CD16-mediated p21ras activation is associated with Shc and p36 tyrosine phosphorylation and their binding with Grb2 in human natural killer cells

PMCID: PMC2192421  PMID: 8551221

Abstract

The Src homology (SH) 2/SH3 domain-containing protein Grb2 and the oncoprotein Shc have been implicated in a highly conserved mechanism that regulates p21ras activation. We investigated the involvement of these adaptor proteins in the signaling pathway induced by CD16 or interleukin (IL) 2R triggering in human natural killer (NK) cells. Both p46 and p52 forms of Shc were rapidly and transiently tyrosine phosphorylated upon CD16 or IL-2 stimulation with different kinetics. Shc immunoprecipitates from lysates of CD16- or IL-2-stimulated NK cells contained Grb2 and an unidentified 145-kD tyrosine phosphoprotein. Grb2 immunoprecipitates from anti-CD16-stimulated NK cells contained not only Shc, but also a 36-kD tyrosine phosphoprotein (p36). The interaction between Grb2 and Shc or p36 occurred via the Grb2SH2 domain as indicated by in vitro binding assays using a bacteriologically synthesized glutathione S-transferase-Grb2SH2 fusion protein. We also present evidence that p21ras is activated by CD16 and IL-2R cross-linking. Accumulation of guanosine triphosphate-bound Ras was detected within 1 minute and occurred with kinetics similar to inductive protein tyrosine phosphorylation and Grb2 association of Shc and p36 adaptor proteins.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  1. Azzoni L., Kamoun M., Salcedo T. W., Kanakaraj P., Perussia B. Stimulation of Fc gamma RIIIA results in phospholipase C-gamma 1 tyrosine phosphorylation and p56lck activation. J Exp Med. 1992 Dec 1;176(6):1745–1750. doi: 10.1084/jem.176.6.1745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balboa M. A., Balsinde J., Aramburu J., Mollinedo F., López-Botet M. Phospholipase D activation in human natural killer cells through the Kp43 and CD16 surface antigens takes place by different mechanisms. Involvement of the phospholipase D pathway in tumor necrosis factor alpha synthesis. J Exp Med. 1992 Jul 1;176(1):9–17. doi: 10.1084/jem.176.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baldari C. T., Pelicci G., Di Somma M. M., Milia E., Giuli S., Pelicci P. G., Telford J. L. Inhibition of CD4/p56lck signaling by a dominant negative mutant of the Shc adaptor protein. Oncogene. 1995 Mar 16;10(6):1141–1147. [PubMed] [Google Scholar]
  4. Basu T., Warne P. H., Downward J. Role of Shc in the activation of Ras in response to epidermal growth factor and nerve growth factor. Oncogene. 1994 Dec;9(12):3483–3491. [PubMed] [Google Scholar]
  5. Bonnema J. D., Karnitz L. M., Schoon R. A., Abraham R. T., Leibson P. J. Fc receptor stimulation of phosphatidylinositol 3-kinase in natural killer cells is associated with protein kinase C-independent granule release and cell-mediated cytotoxicity. J Exp Med. 1994 Oct 1;180(4):1427–1435. doi: 10.1084/jem.180.4.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borrego F., Peña J., Solana R. Regulation of CD69 expression on human natural killer cells: differential involvement of protein kinase C and protein tyrosine kinases. Eur J Immunol. 1993 May;23(5):1039–1043. doi: 10.1002/eji.1830230509. [DOI] [PubMed] [Google Scholar]
  7. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  8. Bowtell D., Fu P., Simon M., Senior P. Identification of murine homologues of the Drosophila son of sevenless gene: potential activators of ras. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6511–6515. doi: 10.1073/pnas.89.14.6511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Buday L., Egan S. E., Rodriguez Viciana P., Cantrell D. A., Downward J. A complex of Grb2 adaptor protein, Sos exchange factor, and a 36-kDa membrane-bound tyrosine phosphoprotein is implicated in ras activation in T cells. J Biol Chem. 1994 Mar 25;269(12):9019–9023. [PubMed] [Google Scholar]
  10. Burns L. A., Karnitz L. M., Sutor S. L., Abraham R. T. Interleukin-2-induced tyrosine phosphorylation of p52shc in T lymphocytes. J Biol Chem. 1993 Aug 25;268(24):17659–17661. [PubMed] [Google Scholar]
  11. Cassatella M. A., Anegón I., Cuturi M. C., Griskey P., Trinchieri G., Perussia B. Fc gamma R(CD16) interaction with ligand induces Ca2+ mobilization and phosphoinositide turnover in human natural killer cells. Role of Ca2+ in Fc gamma R(CD16)-induced transcription and expression of lymphokine genes. J Exp Med. 1989 Feb 1;169(2):549–567. doi: 10.1084/jem.169.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. D'Ambrosio D., Cantrell D. A., Frati L., Santoni A., Testi R. Involvement of p21ras activation in T cell CD69 expression. Eur J Immunol. 1994 Mar;24(3):616–620. doi: 10.1002/eji.1830240319. [DOI] [PubMed] [Google Scholar]
  13. Downward J., Graves J. D., Warne P. H., Rayter S., Cantrell D. A. Stimulation of p21ras upon T-cell activation. Nature. 1990 Aug 23;346(6286):719–723. doi: 10.1038/346719a0. [DOI] [PubMed] [Google Scholar]
  14. Downward J. Regulatory mechanisms for ras proteins. Bioessays. 1992 Mar;14(3):177–184. doi: 10.1002/bies.950140308. [DOI] [PubMed] [Google Scholar]
  15. Downward J. The GRB2/Sem-5 adaptor protein. FEBS Lett. 1994 Jan 31;338(2):113–117. doi: 10.1016/0014-5793(94)80346-3. [DOI] [PubMed] [Google Scholar]
  16. Egan S. E., Weinberg R. A. The pathway to signal achievement. Nature. 1993 Oct 28;365(6449):781–783. doi: 10.1038/365781a0. [DOI] [PubMed] [Google Scholar]
  17. Gilliland L. K., Schieven G. L., Norris N. A., Kanner S. B., Aruffo A., Ledbetter J. A. Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains. J Biol Chem. 1992 Jul 5;267(19):13610–13616. [PubMed] [Google Scholar]
  18. Gismondi A., Milella M., Palmieri G., Piccoli M., Frati L., Santoni A. Stimulation of protein tyrosine phosphorylation by interaction of NK cells with fibronectin via alpha 4 beta 1 and alpha 5 beta 1. J Immunol. 1995 Apr 1;154(7):3128–3137. [PubMed] [Google Scholar]
  19. Hatakeyama M., Kono T., Kobayashi N., Kawahara A., Levin S. D., Perlmutter R. M., Taniguchi T. Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association. Science. 1991 Jun 14;252(5012):1523–1528. doi: 10.1126/science.2047859. [DOI] [PubMed] [Google Scholar]
  20. Hill C. S., Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell. 1995 Jan 27;80(2):199–211. doi: 10.1016/0092-8674(95)90403-4. [DOI] [PubMed] [Google Scholar]
  21. Kanakaraj P., Duckworth B., Azzoni L., Kamoun M., Cantley L. C., Perussia B. Phosphatidylinositol-3 kinase activation induced upon Fc gamma RIIIA-ligand interaction. J Exp Med. 1994 Feb 1;179(2):551–558. doi: 10.1084/jem.179.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kavanaugh W. M., Williams L. T. An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science. 1994 Dec 16;266(5192):1862–1865. doi: 10.1126/science.7527937. [DOI] [PubMed] [Google Scholar]
  23. Lanier L. L., Buck D. W., Rhodes L., Ding A., Evans E., Barney C., Phillips J. H. Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 activation antigen. J Exp Med. 1988 May 1;167(5):1572–1585. doi: 10.1084/jem.167.5.1572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lankester A. C., van Schijndel G. M., Rood P. M., Verhoeven A. J., van Lier R. A. B cell antigen receptor cross-linking induces tyrosine phosphorylation and membrane translocation of a multimeric Shc complex that is augmented by CD19 co-ligation. Eur J Immunol. 1994 Nov;24(11):2818–2825. doi: 10.1002/eji.1830241136. [DOI] [PubMed] [Google Scholar]
  25. Lin J. H., Wu L. S., Tsai K. T., Leu S. P., Jeang Y. F., Hsieh M. T. Effects of ginseng on the blood chemistry profile of dexamethasone-treated male rats. Am J Chin Med. 1995;23(2):167–172. doi: 10.1142/S0192415X95000225. [DOI] [PubMed] [Google Scholar]
  26. Liu L., Damen J. E., Cutler R. L., Krystal G. Multiple cytokines stimulate the binding of a common 145-kilodalton protein to Shc at the Grb2 recognition site of Shc. Mol Cell Biol. 1994 Oct;14(10):6926–6935. doi: 10.1128/mcb.14.10.6926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. McGlade J., Cheng A., Pelicci G., Pelicci P. G., Pawson T. Shc proteins are phosphorylated and regulated by the v-Src and v-Fps protein-tyrosine kinases. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8869–8873. doi: 10.1073/pnas.89.19.8869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. O'Shea J. J., Weissman A. M., Kennedy I. C., Ortaldo J. R. Engagement of the natural killer cell IgG Fc receptor results in tyrosine phosphorylation of the zeta chain. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):350–354. doi: 10.1073/pnas.88.2.350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pelicci G., Lanfrancone L., Grignani F., McGlade J., Cavallo F., Forni G., Nicoletti I., Grignani F., Pawson T., Pelicci P. G. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell. 1992 Jul 10;70(1):93–104. doi: 10.1016/0092-8674(92)90536-l. [DOI] [PubMed] [Google Scholar]
  31. Perussia B., Trinchieri G., Jackson A., Warner N. L., Faust J., Rumpold H., Kraft D., Lanier L. L. The Fc receptor for IgG on human natural killer cells: phenotypic, functional, and comparative studies with monoclonal antibodies. J Immunol. 1984 Jul;133(1):180–189. [PubMed] [Google Scholar]
  32. Pronk G. J., de Vries-Smits A. M., Buday L., Downward J., Maassen J. A., Medema R. H., Bos J. L. Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras. Mol Cell Biol. 1994 Mar;14(3):1575–1581. doi: 10.1128/mcb.14.3.1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ravetch J. V., Kinet J. P. Fc receptors. Annu Rev Immunol. 1991;9:457–492. doi: 10.1146/annurev.iy.09.040191.002325. [DOI] [PubMed] [Google Scholar]
  34. Ravichandran K. S., Burakoff S. J. The adapter protein Shc interacts with the interleukin-2 (IL-2) receptor upon IL-2 stimulation. J Biol Chem. 1994 Jan 21;269(3):1599–1602. [PubMed] [Google Scholar]
  35. Ravichandran K. S., Lee K. K., Songyang Z., Cantley L. C., Burn P., Burakoff S. J. Interaction of Shc with the zeta chain of the T cell receptor upon T cell activation. Science. 1993 Nov 5;262(5135):902–905. doi: 10.1126/science.8235613. [DOI] [PubMed] [Google Scholar]
  36. Ravichandran K. S., Lorenz U., Shoelson S. E., Burakoff S. J. Interaction of Shc with Grb2 regulates association of Grb2 with mSOS. Mol Cell Biol. 1995 Feb;15(2):593–600. doi: 10.1128/mcb.15.2.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rayter S. I., Woodrow M., Lucas S. C., Cantrell D. A., Downward J. p21ras mediates control of IL-2 gene promoter function in T cell activation. EMBO J. 1992 Dec;11(12):4549–4556. doi: 10.1002/j.1460-2075.1992.tb05556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rodewald H. R., Moingeon P., Lucich J. L., Dosiou C., Lopez P., Reinherz E. L. A population of early fetal thymocytes expressing Fc gamma RII/III contains precursors of T lymphocytes and natural killer cells. Cell. 1992 Apr 3;69(1):139–150. doi: 10.1016/0092-8674(92)90125-v. [DOI] [PubMed] [Google Scholar]
  39. Rudd C. E., Trevillyan J. M., Dasgupta J. D., Wong L. L., Schlossman S. F. The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5190–5194. doi: 10.1073/pnas.85.14.5190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Salcedo T. W., Kurosaki T., Kanakaraj P., Ravetch J. V., Perussia B. Physical and functional association of p56lck with Fc gamma RIIIA (CD16) in natural killer cells. J Exp Med. 1993 May 1;177(5):1475–1480. doi: 10.1084/jem.177.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sasaoka T., Draznin B., Leitner J. W., Langlois W. J., Olefsky J. M. Shc is the predominant signaling molecule coupling insulin receptors to activation of guanine nucleotide releasing factor and p21ras-GTP formation. J Biol Chem. 1994 Apr 8;269(14):10734–10738. [PubMed] [Google Scholar]
  42. Sasaoka T., Langlois W. J., Leitner J. W., Draznin B., Olefsky J. M. The signaling pathway coupling epidermal growth factor receptors to activation of p21ras. J Biol Chem. 1994 Dec 23;269(51):32621–32625. [PubMed] [Google Scholar]
  43. Saxton T. M., van Oostveen I., Bowtell D., Aebersold R., Gold M. R. B cell antigen receptor cross-linking induces phosphorylation of the p21ras oncoprotein activators SHC and mSOS1 as well as assembly of complexes containing SHC, GRB-2, mSOS1, and a 145-kDa tyrosine-phosphorylated protein. J Immunol. 1994 Jul 15;153(2):623–636. [PubMed] [Google Scholar]
  44. Sieh M., Batzer A., Schlessinger J., Weiss A. GRB2 and phospholipase C-gamma 1 associate with a 36- to 38-kilodalton phosphotyrosine protein after T-cell receptor stimulation. Mol Cell Biol. 1994 Jul;14(7):4435–4442. doi: 10.1128/mcb.14.7.4435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Skolnik E. Y., Lee C. H., Batzer A., Vicentini L. M., Zhou M., Daly R., Myers M. J., Jr, Backer J. M., Ullrich A., White M. F. The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. EMBO J. 1993 May;12(5):1929–1936. doi: 10.1002/j.1460-2075.1993.tb05842.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Smit L., de Vries-Smits A. M., Bos J. L., Borst J. B cell antigen receptor stimulation induces formation of a Shc-Grb2 complex containing multiple tyrosine-phosphorylated proteins. J Biol Chem. 1994 Aug 12;269(32):20209–20212. [PubMed] [Google Scholar]
  47. Sánchez M. J., Muench M. O., Roncarolo M. G., Lanier L. L., Phillips J. H. Identification of a common T/natural killer cell progenitor in human fetal thymus. J Exp Med. 1994 Aug 1;180(2):569–576. doi: 10.1084/jem.180.2.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ting A. T., Karnitz L. M., Schoon R. A., Abraham R. T., Leibson P. J. Fc gamma receptor activation induces the tyrosine phosphorylation of both phospholipase C (PLC)-gamma 1 and PLC-gamma 2 in natural killer cells. J Exp Med. 1992 Dec 1;176(6):1751–1755. doi: 10.1084/jem.176.6.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Trinchieri G. Biology of natural killer cells. Adv Immunol. 1989;47:187–376. doi: 10.1016/S0065-2776(08)60664-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Veillette A., Bookman M. A., Horak E. M., Bolen J. B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 1988 Oct 21;55(2):301–308. doi: 10.1016/0092-8674(88)90053-0. [DOI] [PubMed] [Google Scholar]
  51. Vivier E., Morin P., O'Brien C., Druker B., Schlossman S. F., Anderson P. Tyrosine phosphorylation of the Fc gamma RIII(CD16): zeta complex in human natural killer cells. Induction by antibody-dependent cytotoxicity but not by natural killing. J Immunol. 1991 Jan 1;146(1):206–210. [PubMed] [Google Scholar]
  52. Weng Z., Thomas S. M., Rickles R. J., Taylor J. A., Brauer A. W., Seidel-Dugan C., Michael W. M., Dreyfuss G., Brugge J. S. Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains. Mol Cell Biol. 1994 Jul;14(7):4509–4521. doi: 10.1128/mcb.14.7.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. van Noesel C. J., Lankester A. C., van Schijndel G. M., van Lier R. A. The CR2/CD19 complex on human B cells contains the src-family kinase Lyn. Int Immunol. 1993 Jul;5(7):699–705. doi: 10.1093/intimm/5.7.699. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES