Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Jul;15(7):3571–3578. doi: 10.1128/mcb.15.7.3571

Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells.

H Meisner 1, B R Conway 1, D Hartley 1, M P Czech 1
PMCID: PMC230594  PMID: 7791764

Abstract

T-cell receptor (TCR) cross-linking increases tyrosine phosphorylation of multiple proteins, only a few of which have been identified. One of the most rapidly tyrosine-phosphorylated polypeptides is the 120-kDa product of the proto-oncogene c-cbl, a cytosolic and cytoskeletal protein containing multiple proline-rich motifs that are potential binding sites for proteins containing Src homology 3 (SH3) domains. We report here that in cultured Jurkat T cells, Cbl is coprecipitated with antibody against the adapter protein Grb2. Upon activation of Jurkat T cells via the TCR-CD3 complex, we find that high-affinity binding of Cbl requires the N-terminal SH3 domain of GST-Grb2 fusion protein but after cross-linking of the TCR-CD3 and CD4 receptors, Cbl binds equally to its SH2 domain. Grb2 antisera also precipitated p85 from serum-starved cells, while TCR activation increased p85 and tyrosine-phosphorylated Cbl but not Cbl protein in Grb2 immunocomplexes. Phosphatidylinositol (PI) 3-kinase activity was immunoprecipitated from serum-starved cells with Cbl and to a lesser extent with Grb2 antisera, and TCR cross-linking increased this activity severalfold. The PI 3-kinase activity associated with Cbl amounted to 5 to 10% of the total cellular activity that could be precipitated by p85 antisera. The Ras exchange factor Son-of-sevenless 1 (Sos-1) was not found in anti-Cbl immunoprecipitates from activated cells, and Cbl was not detectable in anti-Sos-1 precipitates, supporting the likelihood that Sos-Grb2 and Cbl-Grb2 are present as distinct complexes. Taken together, these data suggest that Cbl function in Jurkat T cells involves its constitutive association with Grb2 and its recruitment of PI 3-kinase in response to TCR activation.

Full Text

The Full Text of this article is available as a PDF (379.4 KB).

Selected References

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

  1. Andoniou C. E., Thien C. B., Langdon W. Y. Tumour induction by activated abl involves tyrosine phosphorylation of the product of the cbl oncogene. EMBO J. 1994 Oct 3;13(19):4515–4523. doi: 10.1002/j.1460-2075.1994.tb06773.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auger K. R., Serunian L. A., Soltoff S. P., Libby P., Cantley L. C. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989 Apr 7;57(1):167–175. doi: 10.1016/0092-8674(89)90182-7. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Baltensperger K., Kozma L. M., Cherniack A. D., Klarlund J. K., Chawla A., Banerjee U., Czech M. P. Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes. Science. 1993 Jun 25;260(5116):1950–1952. doi: 10.1126/science.8391166. [DOI] [PubMed] [Google Scholar]
  5. Batzer A. G., Rotin D., Ureña J. M., Skolnik E. Y., Schlessinger J. Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol Cell Biol. 1994 Aug;14(8):5192–5201. doi: 10.1128/mcb.14.8.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blake T. J., Heath K. G., Langdon W. Y. The truncation that generated the v-cbl oncogene reveals an ability for nuclear transport, DNA binding and acute transformation. EMBO J. 1993 May;12(5):2017–2026. doi: 10.1002/j.1460-2075.1993.tb05851.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blake T. J., Shapiro M., Morse H. C., 3rd, Langdon W. Y. The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif. Oncogene. 1991 Apr;6(4):653–657. [PubMed] [Google Scholar]
  8. Couture C., Baier G., Altman A., Mustelin T. p56lck-independent activation and tyrosine phosphorylation of p72syk by T-cell antigen receptor/CD3 stimulation. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5301–5305. doi: 10.1073/pnas.91.12.5301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donovan J. A., Wange R. L., Langdon W. Y., Samelson L. E. The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor. J Biol Chem. 1994 Sep 16;269(37):22921–22924. [PubMed] [Google Scholar]
  10. 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]
  11. Exley M., Varticovski L., Peter M., Sancho J., Terhorst C. Association of phosphatidylinositol 3-kinase with a specific sequence of the T cell receptor zeta chain is dependent on T cell activation. J Biol Chem. 1994 May 27;269(21):15140–15146. [PubMed] [Google Scholar]
  12. Ghosh S., Gifford A. M., Riviere L. R., Tempst P., Nolan G. P., Baltimore D. Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal. Cell. 1990 Sep 7;62(5):1019–1029. doi: 10.1016/0092-8674(90)90276-k. [DOI] [PubMed] [Google Scholar]
  13. Herman P. K., Emr S. D. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6742–6754. doi: 10.1128/mcb.10.12.6742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Howe L. R., Weiss A. Multiple kinases mediate T-cell-receptor signaling. Trends Biochem Sci. 1995 Feb;20(2):59–64. doi: 10.1016/s0968-0004(00)88958-6. [DOI] [PubMed] [Google Scholar]
  15. Joly M., Kazlauskas A., Fay F. S., Corvera S. Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites. Science. 1994 Feb 4;263(5147):684–687. doi: 10.1126/science.8303278. [DOI] [PubMed] [Google Scholar]
  16. June C. H., Fletcher M. C., Ledbetter J. A., Samelson L. E. Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation. J Immunol. 1990 Mar 1;144(5):1591–1599. [PubMed] [Google Scholar]
  17. Kapeller R., Cantley L. C. Phosphatidylinositol 3-kinase. Bioessays. 1994 Aug;16(8):565–576. doi: 10.1002/bies.950160810. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Mayer B. J., Baltimore D. Signalling through SH2 and SH3 domains. Trends Cell Biol. 1993 Jan;3(1):8–13. doi: 10.1016/0962-8924(93)90194-6. [DOI] [PubMed] [Google Scholar]
  20. Motto D. G., Ross S. E., Jackman J. K., Sun Q., Olson A. L., Findell P. R., Koretzky G. A. In vivo association of Grb2 with pp116, a substrate of the T cell antigen receptor-activated protein tyrosine kinase. J Biol Chem. 1994 Aug 26;269(34):21608–21613. [PubMed] [Google Scholar]
  21. Mustelin T., Burn P. Regulation of src family tyrosine kinases in lymphocytes. Trends Biochem Sci. 1993 Jun;18(6):215–220. doi: 10.1016/0968-0004(93)90192-p. [DOI] [PubMed] [Google Scholar]
  22. Myers M. G., Jr, Sun X. J., White M. F. The IRS-1 signaling system. Trends Biochem Sci. 1994 Jul;19(7):289–293. doi: 10.1016/0968-0004(94)90007-8. [DOI] [PubMed] [Google Scholar]
  23. Ninomiya N., Hazeki K., Fukui Y., Seya T., Okada T., Hazeki O., Ui M. Involvement of phosphatidylinositol 3-kinase in Fc gamma receptor signaling. J Biol Chem. 1994 Sep 9;269(36):22732–22737. [PubMed] [Google Scholar]
  24. Panchamoorthy G., Fukazawa T., Stolz L., Payne G., Reedquist K., Shoelson S., Songyang Z., Cantley L., Walsh C., Band H. Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn. Mol Cell Biol. 1994 Sep;14(9):6372–6385. doi: 10.1128/mcb.14.9.6372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pawson T., Gish G. D. SH2 and SH3 domains: from structure to function. Cell. 1992 Oct 30;71(3):359–362. doi: 10.1016/0092-8674(92)90504-6. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Prasad K. V., Janssen O., Kapeller R., Raab M., Cantley L. C., Rudd C. E. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7366–7370. doi: 10.1073/pnas.90.15.7366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Reedquist K. A., Fukazawa T., Druker B., Panchamoorthy G., Shoelson S. E., Band H. Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4135–4139. doi: 10.1073/pnas.91.10.4135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reif K., Buday L., Downward J., Cantrell D. A. SH3 domains of the adapter molecule Grb2 complex with two proteins in T cells: the guanine nucleotide exchange protein Sos and a 75-kDa protein that is a substrate for T cell antigen receptor-activated tyrosine kinases. J Biol Chem. 1994 May 13;269(19):14081–14087. [PubMed] [Google Scholar]
  31. Reth M. Antigen receptor tail clue. Nature. 1989 Mar 30;338(6214):383–384. doi: 10.1038/338383b0. [DOI] [PubMed] [Google Scholar]
  32. Rivero-Lezcano O. M., Sameshima J. H., Marcilla A., Robbins K. C. Physical association between Src homology 3 elements and the protein product of the c-cbl proto-oncogene. J Biol Chem. 1994 Jul 1;269(26):17363–17366. [PubMed] [Google Scholar]
  33. Rozakis-Adcock M., McGlade J., Mbamalu G., Pelicci G., Daly R., Li W., Batzer A., Thomas S., Brugge J., Pelicci P. G. Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature. 1992 Dec 17;360(6405):689–692. doi: 10.1038/360689a0. [DOI] [PubMed] [Google Scholar]
  34. Rudd C. E., Janssen O., Cai Y. C., da Silva A. J., Raab M., Prasad K. V. Two-step TCR zeta/CD3-CD4 and CD28 signaling in T cells: SH2/SH3 domains, protein-tyrosine and lipid kinases. Immunol Today. 1994 May;15(5):225–234. doi: 10.1016/0167-5699(94)90248-8. [DOI] [PubMed] [Google Scholar]
  35. Samelson L. E., Klausner R. D. Tyrosine kinases and tyrosine-based activation motifs. Current research on activation via the T cell antigen receptor. J Biol Chem. 1992 Dec 15;267(35):24913–24916. [PubMed] [Google Scholar]
  36. Samelson L. E., Phillips A. F., Luong E. T., Klausner R. D. Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4358–4362. doi: 10.1073/pnas.87.11.4358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Seedorf K., Kostka G., Lammers R., Bashkin P., Daly R., Burgess W. H., van der Bliek A. M., Schlessinger J., Ullrich A. Dynamin binds to SH3 domains of phospholipase C gamma and GRB-2. J Biol Chem. 1994 Jun 10;269(23):16009–16014. [PubMed] [Google Scholar]
  38. 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]
  39. Songyang Z., Shoelson S. E., McGlade J., Olivier P., Pawson T., Bustelo X. R., Barbacid M., Sabe H., Hanafusa H., Yi T. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol Cell Biol. 1994 Apr;14(4):2777–2785. doi: 10.1128/mcb.14.4.2777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Straus D. B., Weiss A. The CD3 chains of the T cell antigen receptor associate with the ZAP-70 tyrosine kinase and are tyrosine phosphorylated after receptor stimulation. J Exp Med. 1993 Nov 1;178(5):1523–1530. doi: 10.1084/jem.178.5.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tanaka S., Morishita T., Hashimoto Y., Hattori S., Nakamura S., Shibuya M., Matuoka K., Takenawa T., Kurata T., Nagashima K. C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3443–3447. doi: 10.1073/pnas.91.8.3443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vogel L. B., Fujita D. J. The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3'-kinase from T lymphocytes. Mol Cell Biol. 1993 Dec;13(12):7408–7417. doi: 10.1128/mcb.13.12.7408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang L. M., Keegan A. D., Li W., Lienhard G. E., Pacini S., Gutkind J. S., Myers M. G., Jr, Sun X. J., White M. F., Aaronson S. A. Common elements in interleukin 4 and insulin signaling pathways in factor-dependent hematopoietic cells. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4032–4036. doi: 10.1073/pnas.90.9.4032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ward S. G., Ley S. C., MacPhee C., Cantrell D. A. Regulation of D-3 phosphoinositides during T cell activation via the T cell antigen receptor/CD3 complex and CD2 antigens. Eur J Immunol. 1992 Jan;22(1):45–49. doi: 10.1002/eji.1830220108. [DOI] [PubMed] [Google Scholar]
  45. Weiss A., Imboden J. B. Cell surface molecules and early events involved in human T lymphocyte activation. Adv Immunol. 1987;41:1–38. doi: 10.1016/s0065-2776(08)60029-2. [DOI] [PubMed] [Google Scholar]
  46. Weiss A., Littman D. R. Signal transduction by lymphocyte antigen receptors. Cell. 1994 Jan 28;76(2):263–274. doi: 10.1016/0092-8674(94)90334-4. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES