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. 1996 Nov 1;184(5):1713–1723. doi: 10.1084/jem.184.5.1713

Characterization of Cbl tyrosine phosphorylation and a Cbl-Syk complex in RBL-2H3 cells

PMCID: PMC2192902  PMID: 8920860

Abstract

Tyrosine phosphorylation of the Cbl protooncogene has been shown to occur after engagement of a number of different receptors on hematopoietic cells. However, the mechanisms by which these receptors induce Cbl tyrosine phosphorylation are poorly understood. Here we demonstrate that engagement of the high affinity IgE receptor (Fc epsilon R1) leads to the tyrosine phosphorylation of Cbl and analyze how this occurs. We show that at least part of Fc epsilon R1-induced Cbl tyrosine phosphorylation is mediated by the Syk tyrosine kinase, and that the Syk-dependent tyrosine phosphorylation of Cbl occurs mainly distal to the Cbl proline-rich region within the COOH-terminal 250 amino acids. Furthermore, we show by coprecipitation that Cbl is present in a complex with Syk before receptor engagement, that the proline-rich region of Cbl and a region of Syk comprised of the two SH2 domains and intradomain linker are required for formation of the complex, and that little or no tyrosine-phosphorylated Cbl is detected in complex with Syk. Overexpression of truncation mutants of Cbl capable of binding Syk has the effect of blocking tyrosine phosphorylation of endogenous Cbl. These results define a potentially important intramolecular interaction in mast cells and suggest a complex function for Cbl in intracellular signaling pathways.

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Selected References

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  1. Adamczewski M., Kinet J. P. The high-affinity receptor for immunoglobulin E. Chem Immunol. 1994;59:173–190. [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Bowtell D. D., Langdon W. Y. The protein product of the c-cbl oncogene rapidly complexes with the EGF receptor and is tyrosine phosphorylated following EGF stimulation. Oncogene. 1995 Oct 19;11(8):1561–1567. [PubMed] [Google Scholar]
  5. Buday L., Khwaja A., Sipeki S., Faragó A., Downward J. Interactions of Cbl with two adapter proteins, Grb2 and Crk, upon T cell activation. J Biol Chem. 1996 Mar 15;271(11):6159–6163. doi: 10.1074/jbc.271.11.6159. [DOI] [PubMed] [Google Scholar]
  6. Cambier J. C. Antigen and Fc receptor signaling. The awesome power of the immunoreceptor tyrosine-based activation motif (ITAM). J Immunol. 1995 Oct 1;155(7):3281–3285. [PubMed] [Google Scholar]
  7. Cambier J. C., Pleiman C. M., Clark M. R. Signal transduction by the B cell antigen receptor and its coreceptors. Annu Rev Immunol. 1994;12:457–486. doi: 10.1146/annurev.iy.12.040194.002325. [DOI] [PubMed] [Google Scholar]
  8. Chan A. C., Dalton M., Johnson R., Kong G. H., Wang T., Thoma R., Kurosaki T. Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function. EMBO J. 1995 Jun 1;14(11):2499–2508. doi: 10.1002/j.1460-2075.1995.tb07247.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cleghon V., Morrison D. K. Raf-1 interacts with Fyn and Src in a non-phosphotyrosine-dependent manner. J Biol Chem. 1994 Jul 1;269(26):17749–17755. [PubMed] [Google Scholar]
  10. Cory G. O., Lovering R. C., Hinshelwood S., MacCarthy-Morrogh L., Levinsky R. J., Kinnon C. The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 domain of Bruton's tyrosine kinase. J Exp Med. 1995 Aug 1;182(2):611–615. doi: 10.1084/jem.182.2.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Fournel M., Davidson D., Weil R., Veillette A. Association of tyrosine protein kinase Zap-70 with the protooncogene product p120c-cbl in T lymphocytes. J Exp Med. 1996 Jan 1;183(1):301–306. doi: 10.1084/jem.183.1.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fukazawa T., Reedquist K. A., Trub T., Soltoff S., Panchamoorthy G., Druker B., Cantley L., Shoelson S. E., Band H. The SH3 domain-binding T cell tyrosyl phosphoprotein p120. Demonstration of its identity with the c-cbl protooncogene product and in vivo complexes with Fyn, Grb2, and phosphatidylinositol 3-kinase. J Biol Chem. 1995 Aug 11;270(32):19141–19150. doi: 10.1074/jbc.270.32.19141. [DOI] [PubMed] [Google Scholar]
  14. Galisteo M. L., Dikic I., Batzer A. G., Langdon W. Y., Schlessinger J. Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and association with epidermal growth factor (EGF) receptor upon EGF stimulation. J Biol Chem. 1995 Sep 1;270(35):20242–20245. doi: 10.1074/jbc.270.35.20242. [DOI] [PubMed] [Google Scholar]
  15. Hartley D., Meisner H., Corvera S. Specific association of the beta isoform of the p85 subunit of phosphatidylinositol-3 kinase with the proto-oncogene c-cbl. J Biol Chem. 1995 Aug 4;270(31):18260–18263. doi: 10.1074/jbc.270.31.18260. [DOI] [PubMed] [Google Scholar]
  16. Hirasawa N., Scharenberg A., Yamamura H., Beaven M. A., Kinet J. P. A requirement for Syk in the activation of the microtubule-associated protein kinase/phospholipase A2 pathway by Fc epsilon R1 is not shared by a G protein-coupled receptor. J Biol Chem. 1995 May 5;270(18):10960–10967. doi: 10.1074/jbc.270.18.10960. [DOI] [PubMed] [Google Scholar]
  17. Iwashima M., Irving B. A., van Oers N. S., Chan A. C., Weiss A. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science. 1994 Feb 25;263(5150):1136–1139. doi: 10.1126/science.7509083. [DOI] [PubMed] [Google Scholar]
  18. Jouvin M. H., Adamczewski M., Numerof R., Letourneur O., Vallé A., Kinet J. P. Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor. J Biol Chem. 1994 Feb 25;269(8):5918–5925. [PubMed] [Google Scholar]
  19. Kim T. J., Kim Y. T., Pillai S. Association of activated phosphatidylinositol 3-kinase with p120cbl in antigen receptor-ligated B cells. J Biol Chem. 1995 Nov 17;270(46):27504–27509. doi: 10.1074/jbc.270.46.27504. [DOI] [PubMed] [Google Scholar]
  20. Kontani K., Kukimoto I., Nishina H., Hoshino S., Hazeki O., Kanaho Y., Katada T. Tyrosine phosphorylation of the c-cbl proto-oncogene product mediated by cell surface antigen CD38 in HL-60 cells. J Biol Chem. 1996 Jan 19;271(3):1534–1537. doi: 10.1074/jbc.271.3.1534. [DOI] [PubMed] [Google Scholar]
  21. Langdon W. Y., Hartley J. W., Klinken S. P., Ruscetti S. K., Morse H. C., 3rd v-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1168–1172. doi: 10.1073/pnas.86.4.1168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lin S., Cicala C., Scharenberg A. M., Kinet J. P. The Fc(epsilon)RIbeta subunit functions as an amplifier of Fc(epsilon)RIgamma-mediated cell activation signals. Cell. 1996 Jun 28;85(7):985–995. doi: 10.1016/s0092-8674(00)81300-8. [DOI] [PubMed] [Google Scholar]
  23. Mahajan S., Fargnoli J., Burkhardt A. L., Kut S. A., Saouaf S. J., Bolen J. B. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase. Mol Cell Biol. 1995 Oct;15(10):5304–5311. doi: 10.1128/mcb.15.10.5304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Malek S. N., Desiderio S. A cyclin-dependent kinase homologue, p130PITSLRE is a phosphotyrosine-independent SH2 ligand. J Biol Chem. 1994 Dec 30;269(52):33009–33020. [PubMed] [Google Scholar]
  25. Marcilla A., Rivero-Lezcano O. M., Agarwal A., Robbins K. C. Identification of the major tyrosine kinase substrate in signaling complexes formed after engagement of Fc gamma receptors. J Biol Chem. 1995 Apr 21;270(16):9115–9120. doi: 10.1074/jbc.270.16.9115. [DOI] [PubMed] [Google Scholar]
  26. Meisner H., Conway B. R., Hartley D., Czech M. P. Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells. Mol Cell Biol. 1995 Jul;15(7):3571–3578. doi: 10.1128/mcb.15.7.3571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Meisner H., Czech M. P. Coupling of the proto-oncogene product c-Cbl to the epidermal growth factor receptor. J Biol Chem. 1995 Oct 27;270(43):25332–25335. doi: 10.1074/jbc.270.43.25332. [DOI] [PubMed] [Google Scholar]
  28. Muller A. J., Pendergast A. M., Havlik M. H., Puil L., Pawson T., Witte O. N. A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction. Mol Cell Biol. 1992 Nov;12(11):5087–5093. doi: 10.1128/mcb.12.11.5087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nunès J. A., Truneh A., Olive D., Cantrell D. A. Signal transduction by CD28 costimulatory receptor on T cells. B7-1 and B7-2 regulation of tyrosine kinase adaptor molecules. J Biol Chem. 1996 Jan 19;271(3):1591–1598. doi: 10.1074/jbc.271.3.1591. [DOI] [PubMed] [Google Scholar]
  30. Odai H., Sasaki K., Iwamatsu A., Hanazono Y., Tanaka T., Mitani K., Yazaki Y., Hirai H. The proto-oncogene product c-Cbl becomes tyrosine phosphorylated by stimulation with GM-CSF or Epo and constitutively binds to the SH3 domain of Grb2/Ash in human hematopoietic cells. J Biol Chem. 1995 May 5;270(18):10800–10805. doi: 10.1074/jbc.270.18.10800. [DOI] [PubMed] [Google Scholar]
  31. Panchamoorthy G., Fukazawa T., Miyake S., Soltoff S., Reedquist K., Druker B., Shoelson S., Cantley L., Band H. p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase. J Biol Chem. 1996 Feb 9;271(6):3187–3194. doi: 10.1074/jbc.271.6.3187. [DOI] [PubMed] [Google Scholar]
  32. Park I., Chung J., Walsh C. T., Yun Y., Strominger J. L., Shin J. Phosphotyrosine-independent binding of a 62-kDa protein to the src homology 2 (SH2) domain of p56lck and its regulation by phosphorylation of Ser-59 in the lck unique N-terminal region. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12338–12342. doi: 10.1073/pnas.92.26.12338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Qian D., Mollenauer M. N., Weiss A. Dominant-negative zeta-associated protein 70 inhibits T cell antigen receptor signaling. J Exp Med. 1996 Feb 1;183(2):611–620. doi: 10.1084/jem.183.2.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rawlings D. J., Scharenberg A. M., Park H., Wahl M. I., Lin S., Kato R. M., Fluckiger A. C., Witte O. N., Kinet J. P. Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. Science. 1996 Feb 9;271(5250):822–825. doi: 10.1126/science.271.5250.822. [DOI] [PubMed] [Google Scholar]
  35. Ribon V., Hubbell S., Herrera R., Saltiel A. R. The product of the cbl oncogene forms stable complexes in vivo with endogenous Crk in a tyrosine phosphorylation-dependent manner. Mol Cell Biol. 1996 Jan;16(1):45–52. doi: 10.1128/mcb.16.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Rowley R. B., Burkhardt A. L., Chao H. G., Matsueda G. R., Bolen J. B. Syk protein-tyrosine kinase is regulated by tyrosine-phosphorylated Ig alpha/Ig beta immunoreceptor tyrosine activation motif binding and autophosphorylation. J Biol Chem. 1995 May 12;270(19):11590–11594. doi: 10.1074/jbc.270.19.11590. [DOI] [PubMed] [Google Scholar]
  38. Samelson L. E., Donovan J. A., Isakov N., Ota Y., Wange R. L. Signal transduction mediated by the T-cell antigen receptor. Ann N Y Acad Sci. 1995 Sep 7;766:157–172. doi: 10.1111/j.1749-6632.1995.tb26659.x. [DOI] [PubMed] [Google Scholar]
  39. Samelson L. E., Patel M. D., Weissman A. M., Harford J. B., Klausner R. D. Antigen activation of murine T cells induces tyrosine phosphorylation of a polypeptide associated with the T cell antigen receptor. Cell. 1986 Sep 26;46(7):1083–1090. doi: 10.1016/0092-8674(86)90708-7. [DOI] [PubMed] [Google Scholar]
  40. Scharenberg A. M., Kinet J. P. Early events in mast cell signal transduction. Chem Immunol. 1995;61:72–87. [PubMed] [Google Scholar]
  41. Scharenberg A. M., Kinet J. P. Initial events in Fc epsilon RI signal transduction. J Allergy Clin Immunol. 1994 Dec;94(6 Pt 2):1142–1146. doi: 10.1016/0091-6749(94)90323-9. [DOI] [PubMed] [Google Scholar]
  42. Scharenberg A. M., Lin S., Cuenod B., Yamamura H., Kinet J. P. Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering. EMBO J. 1995 Jul 17;14(14):3385–3394. doi: 10.1002/j.1460-2075.1995.tb07344.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shiue L., Zoller M. J., Brugge J. S. Syk is activated by phosphotyrosine-containing peptides representing the tyrosine-based activation motifs of the high affinity receptor for IgE. J Biol Chem. 1995 May 5;270(18):10498–10502. doi: 10.1074/jbc.270.18.10498. [DOI] [PubMed] [Google Scholar]
  44. Soltoff S. P., Cantley L. C. p120cbl is a cytosolic adapter protein that associates with phosphoinositide 3-kinase in response to epidermal growth factor in PC12 and other cells. J Biol Chem. 1996 Jan 5;271(1):563–567. doi: 10.1074/jbc.271.1.563. [DOI] [PubMed] [Google Scholar]
  45. Taniguchi T., Kobayashi T., Kondo J., Takahashi K., Nakamura H., Suzuki J., Nagai K., Yamada T., Nakamura S., Yamamura H. Molecular cloning of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to proteolysis. J Biol Chem. 1991 Aug 25;266(24):15790–15796. [PubMed] [Google Scholar]
  46. Tezuka T., Umemori H., Fusaki N., Yagi T., Takata M., Kurosaki T., Yamamoto T. Physical and functional association of the cbl protooncogen product with an src-family protein tyrosine kinase, p53/56lyn, in the B cell antigen receptor-mediated signaling. J Exp Med. 1996 Feb 1;183(2):675–680. doi: 10.1084/jem.183.2.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang Y., Yeung Y. G., Langdon W. Y., Stanley E. R. c-Cbl is transiently tyrosine-phosphorylated, ubiquitinated, and membrane-targeted following CSF-1 stimulation of macrophages. J Biol Chem. 1996 Jan 5;271(1):17–20. doi: 10.1074/jbc.271.1.17. [DOI] [PubMed] [Google Scholar]
  48. Wange R. L., Guitián R., Isakov N., Watts J. D., Aebersold R., Samelson L. E. Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70. J Biol Chem. 1995 Aug 11;270(32):18730–18733. doi: 10.1074/jbc.270.32.18730. [DOI] [PubMed] [Google Scholar]
  49. Wange R. L., Isakov N., Burke T. R., Jr, Otaka A., Roller P. P., Watts J. D., Aebersold R., Samelson L. E. F2(Pmp)2-TAM zeta 3, a novel competitive inhibitor of the binding of ZAP-70 to the T cell antigen receptor, blocks early T cell signaling. J Biol Chem. 1995 Jan 13;270(2):944–948. doi: 10.1074/jbc.270.2.944. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Yi T. L., Bolen J. B., Ihle J. N. Hematopoietic cells express two forms of lyn kinase differing by 21 amino acids in the amino terminus. Mol Cell Biol. 1991 May;11(5):2391–2398. doi: 10.1128/mcb.11.5.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yoon C. H., Lee J., Jongeward G. D., Sternberg P. W. Similarity of sli-1, a regulator of vulval development in C. elegans, to the mammalian proto-oncogene c-cbl. Science. 1995 Aug 25;269(5227):1102–1105. doi: 10.1126/science.7652556. [DOI] [PubMed] [Google Scholar]
  53. de Jong R., ten Hoeve J., Heisterkamp N., Groffen J. Crkl is complexed with tyrosine-phosphorylated Cbl in Ph-positive leukemia. J Biol Chem. 1995 Sep 15;270(37):21468–21471. doi: 10.1074/jbc.270.37.21468. [DOI] [PubMed] [Google Scholar]

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