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. 1995 Aug;15(8):4149–4157. doi: 10.1128/mcb.15.8.4149

Activation of the high-affinity immunoglobulin E receptor Fc epsilon RI in RBL-2H3 cells is inhibited by Syk SH2 domains.

J A Taylor 1, J L Karas 1, M K Ram 1, O M Green 1, C Seidel-Dugan 1
PMCID: PMC230653  PMID: 7623809

Abstract

Antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E, Fc epsilon RI, results in the activation of multiple signaling pathways, leading to the release of mediators of the allergic response. One of the earliest responses to receptor stimulation is the tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI and the association of the tyrosine kinase Syk with the phosphorylated receptor. This association is mediated by the SH2 domains of Syk and is believed to be critical for activating signaling pathways resulting in mediator release. To examine the importance of the interaction of Syk with Fc epsilon RI in signaling events following receptor activation, we introduced a protein containing the SH2 domains of Syk into streptolysin O-permeabilized RBL-2H3 cells. The Syk SH2 domains completely inhibited degranulation and leukotriene production following receptor aggregation, and they blocked the increase in protein tyrosine phosphorylation observed after receptor activation. Inhibition was specific for Fc epsilon RI-mediated signaling, since degranulation of cells activated by alternative stimuli was not blocked by the Syk SH2 domains. A protein containing a point mutation in the carboxy-terminal SH2 domain which abolishes phosphotyrosine binding was not inhibitory. In addition, inhibition of degranulation was reversed by pretreatment of the SH2 domains with a tyrosine phosphorylated peptide corresponding to the tyrosine-based activation motif found in the gamma subunit of Fc epsilon RI, the nonphosphorylated peptide had no effect. The association of Syk with the tyrosine-phosphorylated gamma subunit of the activated receptor was blocked by the Syk SH2 domains, and deregulation in cells activated by clustering of Syk directly without Fc epsilon RI aggregation was not affected by the Syk SH2 domains. These results demonstrate that the association of Syk with the activated Fc epsilon RI is critical for both early and late events following receptor activation and confirm the key role Syk plays in signaling through the high-affinity IgE receptor.

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Selected References

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  1. Ali H., Collado-Escobar D. M., Beaven M. A. The rise in concentration of free Ca2+ and of pH provides sequential, synergistic signals for secretion in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells. J Immunol. 1989 Oct 15;143(8):2626–2633. [PubMed] [Google Scholar]
  2. Ali H., Cunha-Melo J. R., Beaven M. A. Receptor-mediated release of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate in rat basophilic leukemia RBL-2H3 cells permeabilized with streptolysin O. Biochim Biophys Acta. 1989 Jan 17;1010(1):88–99. doi: 10.1016/0167-4889(89)90188-2. [DOI] [PubMed] [Google Scholar]
  3. Anderson D., Koch C. A., Grey L., Ellis C., Moran M. F., Pawson T. Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors. Science. 1990 Nov 16;250(4983):979–982. doi: 10.1126/science.2173144. [DOI] [PubMed] [Google Scholar]
  4. Arpaia E., Shahar M., Dadi H., Cohen A., Roifman C. M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase. Cell. 1994 Mar 11;76(5):947–958. doi: 10.1016/0092-8674(94)90368-9. [DOI] [PubMed] [Google Scholar]
  5. Atkinson T. P., Kaliner M. A., Hohman R. J. Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors. J Immunol. 1992 Apr 1;148(7):2194–2200. [PubMed] [Google Scholar]
  6. Beaven M. A., Metzger H. Signal transduction by Fc receptors: the Fc epsilon RI case. Immunol Today. 1993 May;14(5):222–226. doi: 10.1016/0167-5699(93)90167-j. [DOI] [PubMed] [Google Scholar]
  7. Benhamou M., Gutkind J. S., Robbins K. C., Siraganian R. P. Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5327–5330. doi: 10.1073/pnas.87.14.5327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Benhamou M., Ryba N. J., Kihara H., Nishikata H., Siraganian R. P. Protein-tyrosine kinase p72syk in high affinity IgE receptor signaling. Identification as a component of pp72 and association with the receptor gamma chain after receptor aggregation. J Biol Chem. 1993 Nov 5;268(31):23318–23324. [PubMed] [Google Scholar]
  9. Bhakdi S., Weller U., Walev I., Martin E., Jonas D., Palmer M. A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med Microbiol Immunol. 1993 Sep;182(4):167–175. doi: 10.1007/BF00219946. [DOI] [PubMed] [Google Scholar]
  10. Blank U., Ra C., Miller L., White K., Metzger H., Kinet J. P. Complete structure and expression in transfected cells of high affinity IgE receptor. Nature. 1989 Jan 12;337(6203):187–189. doi: 10.1038/337187a0. [DOI] [PubMed] [Google Scholar]
  11. Chan A. C., Irving B. A., Fraser J. D., Weiss A. The zeta chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9166–9170. doi: 10.1073/pnas.88.20.9166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chan A. C., Iwashima M., Turck C. W., Weiss A. ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain. Cell. 1992 Nov 13;71(4):649–662. doi: 10.1016/0092-8674(92)90598-7. [DOI] [PubMed] [Google Scholar]
  13. Chan A. C., Kadlecek T. A., Elder M. E., Filipovich A. H., Kuo W. L., Iwashima M., Parslow T. G., Weiss A. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science. 1994 Jun 10;264(5165):1599–1601. doi: 10.1126/science.8202713. [DOI] [PubMed] [Google Scholar]
  14. Connelly P. A., Farrell C. A., Merenda J. M., Conklyn M. J., Showell H. J. Tyrosine phosphorylation is an early signaling event common to Fc receptor crosslinking in human neutrophils and rat basophilic leukemia cells (RBL-2H3). Biochem Biophys Res Commun. 1991 May 31;177(1):192–201. doi: 10.1016/0006-291x(91)91967-h. [DOI] [PubMed] [Google Scholar]
  15. Cunha-Melo J. R., Gonzaga H. M., Ali H., Huang F. L., Huang K. P., Beaven M. A. Studies of protein kinase C in the rat basophilic leukemia (RBL-2H3) cell reveal that antigen-induced signals are not mimicked by the actions of phorbol myristate acetate and Ca2+ ionophore. J Immunol. 1989 Oct 15;143(8):2617–2625. [PubMed] [Google Scholar]
  16. Eiseman E., Bolen J. B. Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases. Nature. 1992 Jan 2;355(6355):78–80. doi: 10.1038/355078a0. [DOI] [PubMed] [Google Scholar]
  17. Eiseman E., Bolen J. B. Signal transduction by the cytoplasmic domains of Fc epsilon RI-gamma and TCR-zeta in rat basophilic leukemia cells. J Biol Chem. 1992 Oct 15;267(29):21027–21032. [PubMed] [Google Scholar]
  18. Elder M. E., Lin D., Clever J., Chan A. C., Hope T. J., Weiss A., Parslow T. G. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science. 1994 Jun 10;264(5165):1596–1599. doi: 10.1126/science.8202712. [DOI] [PubMed] [Google Scholar]
  19. Fukamachi H., Kawakami Y., Takei M., Ishizaka T., Ishizaka K., Kawakami T. Association of protein-tyrosine kinase with phospholipase C-gamma 1 in bone marrow-derived mouse mast cells. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9524–9528. doi: 10.1073/pnas.89.20.9524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gauen L. K., Zhu Y., Letourneur F., Hu Q., Bolen J. B., Matis L. A., Klausner R. D., Shaw A. S. Interactions of p59fyn and ZAP-70 with T-cell receptor activation motifs: defining the nature of a signalling motif. Mol Cell Biol. 1994 Jun;14(6):3729–3741. doi: 10.1128/mcb.14.6.3729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hutchcroft J. E., Harrison M. L., Geahlen R. L. Association of the 72-kDa protein-tyrosine kinase PTK72 with the B cell antigen receptor. J Biol Chem. 1992 Apr 25;267(12):8613–8619. [PubMed] [Google Scholar]
  22. Hutchcroft J. E., Harrison M. L., Geahlen R. L. B lymphocyte activation is accompanied by phosphorylation of a 72-kDa protein-tyrosine kinase. J Biol Chem. 1991 Aug 15;266(23):14846–14849. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Kawakami T., Inagaki N., Takei M., Fukamachi H., Coggeshall K. M., Ishizaka K., Ishizaka T. Tyrosine phosphorylation is required for mast cell activation by Fc epsilon RI cross-linking. J Immunol. 1992 Jun 1;148(11):3513–3519. [PubMed] [Google Scholar]
  26. Kawakami Y., Yao L., Miura T., Tsukada S., Witte O. N., Kawakami T. Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc epsilon RI cross-linking. Mol Cell Biol. 1994 Aug;14(8):5108–5113. doi: 10.1128/mcb.14.8.5108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kihara H., Siraganian R. P. Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor. J Biol Chem. 1994 Sep 2;269(35):22427–22432. [PubMed] [Google Scholar]
  28. Kinet J. P. The high-affinity receptor for IgE. Curr Opin Immunol. 1989;2(4):499–505. doi: 10.1016/0952-7915(90)90002-x. [DOI] [PubMed] [Google Scholar]
  29. Koyasu S., Tse A. G., Moingeon P., Hussey R. E., Mildonian A., Hannisian J., Clayton L. K., Reinherz E. L. Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6693–6697. doi: 10.1073/pnas.91.14.6693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lavens S. E., Peachell P. T., Warner J. A. Role of tyrosine kinases in IgE-mediated signal transduction in human lung mast cells and basophils. Am J Respir Cell Mol Biol. 1992 Dec;7(6):637–644. doi: 10.1165/ajrcmb/7.6.637. [DOI] [PubMed] [Google Scholar]
  31. Law C. L., Sidorenko S. P., Chandran K. A., Draves K. E., Chan A. C., Weiss A., Edelhoff S., Disteche C. M., Clark E. A. Molecular cloning of human Syk. A B cell protein-tyrosine kinase associated with the surface immunoglobulin M-B cell receptor complex. J Biol Chem. 1994 Apr 22;269(16):12310–12319. [PubMed] [Google Scholar]
  32. Letourneur F., Klausner R. D. Activation of T cells by a tyrosine kinase activation domain in the cytoplasmic tail of CD3 epsilon. Science. 1992 Jan 3;255(5040):79–82. doi: 10.1126/science.1532456. [DOI] [PubMed] [Google Scholar]
  33. Letourneur F., Klausner R. D. T-cell and basophil activation through the cytoplasmic tail of T-cell-receptor zeta family proteins. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8905–8909. doi: 10.1073/pnas.88.20.8905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Marengere L. E., Songyang Z., Gish G. D., Schaller M. D., Parsons J. T., Stern M. J., Cantley L. C., Pawson T. SH2 domain specificity and activity modified by a single residue. Nature. 1994 Jun 9;369(6480):502–505. doi: 10.1038/369502a0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Minoguchi K., Benhamou M., Swaim W. D., Kawakami Y., Kawakami T., Siraganian R. P. Activation of protein tyrosine kinase p72syk by Fc epsilon RI aggregation in rat basophilic leukemia cells. p72syk is a minor component but the major protein tyrosine kinase of pp72. J Biol Chem. 1994 Jun 17;269(24):16902–16908. [PubMed] [Google Scholar]
  38. Oliver J. M., Burg D. L., Wilson B. S., McLaughlin J. L., Geahlen R. L. Inhibition of mast cell Fc epsilon R1-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol. J Biol Chem. 1994 Nov 25;269(47):29697–29703. [PubMed] [Google Scholar]
  39. Oliver J. M., Seagrave J., Stump R. F., Pfeiffer J. R., Deanin G. G. Signal transduction and cellular response in RBL-2H3 mast cells. Prog Allergy. 1988;42:185–245. [PubMed] [Google Scholar]
  40. Ozawa K., Szallasi Z., Kazanietz M. G., Blumberg P. M., Mischak H., Mushinski J. F., Beaven M. A. Ca(2+)-dependent and Ca(2+)-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells. J Biol Chem. 1993 Jan 25;268(3):1749–1756. [PubMed] [Google Scholar]
  41. Paolini R., Jouvin M. H., Kinet J. P. Phosphorylation and dephosphorylation of the high-affinity receptor for immunoglobulin E immediately after receptor engagement and disengagement. Nature. 1991 Oct 31;353(6347):855–858. doi: 10.1038/353855a0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Reth M. Antigen receptor tail clue. Nature. 1989 Mar 30;338(6214):383–384. doi: 10.1038/338383b0. [DOI] [PubMed] [Google Scholar]
  45. Rivera V. M., Brugge J. S. Clustering of Syk is sufficient to induce tyrosine phosphorylation and release of allergic mediators from rat basophilic leukemia cells. Mol Cell Biol. 1995 Mar;15(3):1582–1590. doi: 10.1128/mcb.15.3.1582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Romeo C., Amiot M., Seed B. Sequence requirements for induction of cytolysis by the T cell antigen/Fc receptor zeta chain. Cell. 1992 Mar 6;68(5):889–897. doi: 10.1016/0092-8674(92)90032-8. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Shakarjian M. P., Eiseman E., Penhallow R. C., Bolen J. B. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition in a rat mast cell line. Impairment of tyrosine kinase-dependent signal transduction and the subsequent degranulation response. J Biol Chem. 1993 Jul 15;268(20):15252–15259. [PubMed] [Google Scholar]
  49. Shiue L., Green J., Green O. M., Karas J. L., Morgenstern J. P., Ram M. K., Taylor M. K., Zoller M. J., Zydowsky L. D., Bolen J. B. Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI. Mol Cell Biol. 1995 Jan;15(1):272–281. doi: 10.1128/mcb.15.1.272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Stevens R. L., Austen K. F. Recent advances in the cellular and molecular biology of mast cells. Immunol Today. 1989 Nov;10(11):381–386. doi: 10.1016/0167-5699(89)90272-7. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Takata M., Sabe H., Hata A., Inazu T., Homma Y., Nukada T., Yamamura H., Kurosaki T. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO J. 1994 Mar 15;13(6):1341–1349. doi: 10.1002/j.1460-2075.1994.tb06387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. Wange R. L., Kong A. N., Samelson L. E. A tyrosine-phosphorylated 70-kDa protein binds a photoaffinity analogue of ATP and associates with both the zeta chain and CD3 components of the activated T cell antigen receptor. J Biol Chem. 1992 Jun 15;267(17):11685–11688. [PubMed] [Google Scholar]
  57. Wange R. L., Malek S. N., Desiderio S., Samelson L. E. Tandem SH2 domains of ZAP-70 bind to T cell antigen receptor zeta and CD3 epsilon from activated Jurkat T cells. J Biol Chem. 1993 Sep 15;268(26):19797–19801. [PubMed] [Google Scholar]
  58. Weiss A. T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases. Cell. 1993 Apr 23;73(2):209–212. doi: 10.1016/0092-8674(93)90221-b. [DOI] [PubMed] [Google Scholar]
  59. Yamada K., Jelsema C. L., Beaven M. A. Certain inhibitors of protein serine/threonine kinases also inhibit tyrosine phosphorylation of phospholipase C gamma 1 and other proteins and reveal distinct roles for tyrosine kinase(s) and protein kinase C in stimulated, rat basophilic RBL-2H3 cells. J Immunol. 1992 Aug 1;149(3):1031–1037. [PubMed] [Google Scholar]
  60. Yamada T., Taniguchi T., Yang C., Yasue S., Saito H., Yamamura H. Association with B-cell-antigen receptor with protein-tyrosine kinase p72syk and activation by engagement of membrane IgM. Eur J Biochem. 1993 Apr 1;213(1):455–459. doi: 10.1111/j.1432-1033.1993.tb17781.x. [DOI] [PubMed] [Google Scholar]

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