Logo of jexpmedHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Exp Med. Nov 1, 1996; 184(5): 1713–1723.
PMCID: PMC2192902

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

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.

Full Text

The Full Text of this article is available as a PDF (1.9M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Weiss A, Littman DR. Signal transduction by lymphocyte antigen receptors. Cell. 1994 Jan 28;76(2):263–274. [PubMed]
  • Samelson LE, Donovan JA, Isakov N, Ota Y, Wange RL. Signal transduction mediated by the T-cell antigen receptor. Ann N Y Acad Sci. 1995 Sep 7;766:157–172. [PubMed]
  • Adamczewski M, Kinet JP. The high-affinity receptor for immunoglobulin E. Chem Immunol. 1994;59:173–190. [PubMed]
  • Cambier JC, Pleiman CM, Clark MR. Signal transduction by the B cell antigen receptor and its coreceptors. Annu Rev Immunol. 1994;12:457–486. [PubMed]
  • Cambier JC. 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]
  • Iwashima M, Irving BA, van Oers NS, Chan AC, Weiss A. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science. 1994 Feb 25;263(5150):1136–1139. [PubMed]
  • Jouvin MH, Adamczewski M, Numerof R, Letourneur O, Vallé A, Kinet JP. 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]
  • Shiue L, Zoller MJ, Brugge JS. 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. [PubMed]
  • Rowley RB, Burkhardt AL, Chao HG, Matsueda GR, Bolen JB. 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. [PubMed]
  • Chan AC, Dalton M, Johnson R, Kong GH, 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. [PMC free article] [PubMed]
  • Wange RL, Guitián R, Isakov N, Watts JD, Aebersold R, Samelson LE. Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70. J Biol Chem. 1995 Aug 11;270(32):18730–18733. [PubMed]
  • Rawlings DJ, Scharenberg AM, Park H, Wahl MI, Lin S, Kato RM, Fluckiger AC, Witte ON, Kinet JP. Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. Science. 1996 Feb 9;271(5250):822–825. [PubMed]
  • Mahajan S, Fargnoli J, Burkhardt AL, Kut SA, Saouaf SJ, Bolen JB. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase. Mol Cell Biol. 1995 Oct;15(10):5304–5311. [PMC free article] [PubMed]
  • Donovan JA, Wange RL, Langdon WY, Samelson LE. 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]
  • Meisner H, Conway BR, Hartley D, Czech MP. Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells. Mol Cell Biol. 1995 Jul;15(7):3571–3578. [PMC free article] [PubMed]
  • Cory GO, Lovering RC, Hinshelwood S, MacCarthy-Morrogh L, Levinsky RJ, 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. [PMC free article] [PubMed]
  • Kim TJ, Kim YT, 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. [PubMed]
  • Marcilla A, Rivero-Lezcano OM, Agarwal A, Robbins KC. 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. [PubMed]
  • Galisteo ML, Dikic I, Batzer AG, Langdon WY, 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. [PubMed]
  • Bowtell DD, Langdon WY. 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]
  • 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. [PubMed]
  • Nunès JA, Truneh A, Olive D, Cantrell DA. 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. [PubMed]
  • 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. [PubMed]
  • Langdon WY, Hartley JW, Klinken SP, Ruscetti SK, Morse HC., 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. [PMC free article] [PubMed]
  • Blake TJ, Shapiro M, Morse HC, 3rd, Langdon WY. 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]
  • 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Fukazawa T, Reedquist KA, Trub T, Soltoff S, Panchamoorthy G, Druker B, Cantley L, Shoelson SE, 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. [PubMed]
  • 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. [PubMed]
  • Meisner H, Czech MP. Coupling of the proto-oncogene product c-Cbl to the epidermal growth factor receptor. J Biol Chem. 1995 Oct 27;270(43):25332–25335. [PubMed]
  • Ribon V, Hubbell S, Herrera R, Saltiel AR. 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. [PMC free article] [PubMed]
  • Rivero-Lezcano OM, Sameshima JH, Marcilla A, Robbins KC. 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]
  • Soltoff SP, Cantley LC. 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. [PubMed]
  • Yoon CH, Lee J, Jongeward GD, Sternberg PW. 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. [PubMed]
  • Scharenberg AM, Kinet JP. Initial events in Fc epsilon RI signal transduction. J Allergy Clin Immunol. 1994 Dec;94(6 Pt 2):1142–1146. [PubMed]
  • Scharenberg AM, Kinet JP. Early events in mast cell signal transduction. Chem Immunol. 1995;61:72–87. [PubMed]
  • Scharenberg AM, Lin S, Cuenod B, Yamamura H, Kinet JP. 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. [PMC free article] [PubMed]
  • Blake TJ, Heath KG, Langdon WY. 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. [PMC free article] [PubMed]
  • 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]
  • Hirasawa N, Scharenberg A, Yamamura H, Beaven MA, Kinet JP. 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. [PubMed]
  • Yi TL, Bolen JB, Ihle JN. 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. [PMC free article] [PubMed]
  • Samelson LE, Patel MD, Weissman AM, Harford JB, Klausner RD. 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. [PubMed]
  • Lin S, Cicala C, Scharenberg AM, Kinet JP. 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Wange RL, Isakov N, Burke TR, Jr, Otaka A, Roller PP, Watts JD, Aebersold R, Samelson LE. 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. [PubMed]
  • Qian D, Mollenauer MN, Weiss A. Dominant-negative zeta-associated protein 70 inhibits T cell antigen receptor signaling. J Exp Med. 1996 Feb 1;183(2):611–620. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Muller AJ, Pendergast AM, Havlik MH, Puil L, Pawson T, Witte ON. A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction. Mol Cell Biol. 1992 Nov;12(11):5087–5093. [PMC free article] [PubMed]
  • Cleghon V, Morrison DK. Raf-1 interacts with Fyn and Src in a non-phosphotyrosine-dependent manner. J Biol Chem. 1994 Jul 1;269(26):17749–17755. [PubMed]
  • Malek SN, 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]
  • Park I, Chung J, Walsh CT, Yun Y, Strominger JL, 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Wang Y, Yeung YG, Langdon WY, Stanley ER. 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. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...