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. 1996 Sep;16(9):4735–4743. doi: 10.1128/mcb.16.9.4735

In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

N Bewarder 1, V Weinrich 1, P Budde 1, D Hartmann 1, H Flaswinkel 1, M Reth 1, J Frey 1
PMCID: PMC231474  PMID: 8756631

Abstract

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.

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Selected References

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  1. Alexandropoulos K., Cheng G., Baltimore D. Proline-rich sequences that bind to Src homology 3 domains with individual specificities. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3110–3114. doi: 10.1073/pnas.92.8.3110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amigorena S., Bonnerot C., Drake J. R., Choquet D., Hunziker W., Guillet J. G., Webster P., Sautes C., Mellman I., Fridman W. H. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. Science. 1992 Jun 26;256(5065):1808–1812. doi: 10.1126/science.1535455. [DOI] [PubMed] [Google Scholar]
  3. Brunati A. M., Donella-Deana A., Ruzzene M., Marin O., Pinna L. A. Site specificity of p72syk protein tyrosine kinase: efficient phosphorylation of motifs recognized by Src homology 2 domains of the Src family. FEBS Lett. 1995 Jun 26;367(2):149–152. doi: 10.1016/0014-5793(95)00555-n. [DOI] [PubMed] [Google Scholar]
  4. Budde P., Bewarder N., Weinrich V., Frey J. Biological functions of human Fc gamma RIIa/Fc gamma RIIc in B cells. Eur J Cell Biol. 1994 Jun;64(1):45–60. [PubMed] [Google Scholar]
  5. Budde P., Bewarder N., Weinrich V., Schulzeck O., Frey J. Tyrosine-containing sequence motifs of the human immunoglobulin G receptors FcRIIb1 and FcRIIb2 essential for endocytosis and regulation of calcium flux in B cells. J Biol Chem. 1994 Dec 2;269(48):30636–30644. [PubMed] [Google Scholar]
  6. Burkhardt A. L., Brunswick M., Bolen J. B., Mond J. J. Anti-immunoglobulin stimulation of B lymphocytes activates src-related protein-tyrosine kinases. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7410–7414. doi: 10.1073/pnas.88.16.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cambier J. C., Johnson S. A. Differential binding activity of ARH1/TAM motifs. Immunol Lett. 1995 Jan;44(2-3):77–80. doi: 10.1016/0165-2478(94)00196-x. [DOI] [PubMed] [Google Scholar]
  8. Campbell K. S., Cambier J. C. B lymphocyte antigen receptors (mIg) are non-covalently associated with a disulfide linked, inducibly phosphorylated glycoprotein complex. EMBO J. 1990 Feb;9(2):441–448. doi: 10.1002/j.1460-2075.1990.tb08129.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Campbell M. A., Sefton B. M. Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases. Mol Cell Biol. 1992 May;12(5):2315–2321. doi: 10.1128/mcb.12.5.2315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cassel D. L., Keller M. A., Surrey S., Schwartz E., Schreiber A. D., Rappaport E. F., McKenzie S. E. Differential expression of Fc gamma RIIA, Fc gamma RIIB and Fc gamma RIIC in hematopoietic cells: analysis of transcripts. Mol Immunol. 1993 Apr;30(5):451–460. doi: 10.1016/0161-5890(93)90113-p. [DOI] [PubMed] [Google Scholar]
  11. Chacko G. W., Duchemin A. M., Coggeshall K. M., Osborne J. M., Brandt J. T., Anderson C. L. Clustering of the platelet Fc gamma receptor induces noncovalent association with the tyrosine kinase p72syk. J Biol Chem. 1994 Dec 23;269(51):32435–32440. [PubMed] [Google Scholar]
  12. Clark M. R., Campbell K. S., Kazlauskas A., Johnson S. A., Hertz M., Potter T. A., Pleiman C., Cambier J. C. The B cell antigen receptor complex: association of Ig-alpha and Ig-beta with distinct cytoplasmic effectors. Science. 1992 Oct 2;258(5079):123–126. doi: 10.1126/science.1439759. [DOI] [PubMed] [Google Scholar]
  13. Clark M. R., Johnson S. A., Cambier J. C. Analysis of Ig-alpha-tyrosine kinase interaction reveals two levels of binding specificity and tyrosine phosphorylated Ig-alpha stimulation of Fyn activity. EMBO J. 1994 Apr 15;13(8):1911–1919. doi: 10.1002/j.1460-2075.1994.tb06460.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. D'Ambrosio D., Hippen K. L., Minskoff S. A., Mellman I., Pani G., Siminovitch K. A., Cambier J. C. Recruitment and activation of PTP1C in negative regulation of antigen receptor signaling by Fc gamma RIIB1. Science. 1995 Apr 14;268(5208):293–297. doi: 10.1126/science.7716523. [DOI] [PubMed] [Google Scholar]
  15. Engelhardt W., Geerds C., Frey J. Distribution, inducibility and biological function of the cloned and expressed human beta Fc receptor II. Eur J Immunol. 1990 Jun;20(6):1367–1377. doi: 10.1002/eji.1830200624. [DOI] [PubMed] [Google Scholar]
  16. Engelhardt W., Gorczytza H., Butterweck A., Mönkemann H., Frey J. Structural requirements of the cytoplasmic domains of the human macrophage Fc gamma receptor IIa and B cell Fc gamma receptor IIb2 for the endocytosis of immune complexes. Eur J Immunol. 1991 Sep;21(9):2227–2238. doi: 10.1002/eji.1830210934. [DOI] [PubMed] [Google Scholar]
  17. Flaswinkel H., Barner M., Reth M. The tyrosine activation motif as a target of protein tyrosine kinases and SH2 domains. Semin Immunol. 1995 Feb;7(1):21–27. doi: 10.1016/1044-5323(95)90004-7. [DOI] [PubMed] [Google Scholar]
  18. Flaswinkel H., Reth M. Dual role of the tyrosine activation motif of the Ig-alpha protein during signal transduction via the B cell antigen receptor. EMBO J. 1994 Jan 1;13(1):83–89. doi: 10.1002/j.1460-2075.1994.tb06237.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ghazizadeh S., Bolen J. B., Fleit H. B. Physical and functional association of Src-related protein tyrosine kinases with Fc gamma RII in monocytic THP-1 cells. J Biol Chem. 1994 Mar 25;269(12):8878–8884. [PubMed] [Google Scholar]
  20. Ghazizadeh S., Bolen J. B., Fleit H. B. Tyrosine phosphorylation and association of Syk with Fc gamma RII in monocytic THP-1 cells. Biochem J. 1995 Jan 15;305(Pt 2):669–674. doi: 10.1042/bj3050669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Greenman J., Tutt A. L., George A. J., Pulford K. A., Stevenson G. T., Glennie M. J. Characterization of a new monoclonal anti-Fc gamma RII antibody, AT10, and its incorporation into a bispecific F(ab')2 derivative for recruitment of cytotoxic effectors. Mol Immunol. 1991 Nov;28(11):1243–1254. doi: 10.1016/0161-5890(91)90011-8. [DOI] [PubMed] [Google Scholar]
  22. Hamada F., Aoki M., Akiyama T., Toyoshima K. Association of immunoglobulin G Fc receptor II with Src-like protein-tyrosine kinase Fgr in neutrophils. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6305–6309. doi: 10.1073/pnas.90.13.6305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hombach J., Lottspeich F., Reth M. Identification of the genes encoding the IgM-alpha and Ig-beta components of the IgM antigen receptor complex by amino-terminal sequencing. Eur J Immunol. 1990 Dec;20(12):2795–2799. doi: 10.1002/eji.1830201239. [DOI] [PubMed] [Google Scholar]
  24. Hombach J., Tsubata T., Leclercq L., Stappert H., Reth M. Molecular components of the B-cell antigen receptor complex of the IgM class. Nature. 1990 Feb 22;343(6260):760–762. doi: 10.1038/343760a0. [DOI] [PubMed] [Google Scholar]
  25. Huang M. M., Indik Z., Brass L. F., Hoxie J. A., Schreiber A. D., Brugge J. S. Activation of Fc gamma RII induces tyrosine phosphorylation of multiple proteins including Fc gamma RII. J Biol Chem. 1992 Mar 15;267(8):5467–5473. [PubMed] [Google Scholar]
  26. Hunter S., Huang M. M., Indik Z. K., Schreiber A. D. Fc gamma RIIA-mediated phagocytosis and receptor phosphorylation in cells deficient in the protein tyrosine kinase Src. Exp Hematol. 1993 Oct;21(11):1492–1497. [PubMed] [Google Scholar]
  27. 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]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. Li Z. H., Mahajan S., Prendergast M. M., Fargnoli J., Zhu X., Klages S., Adam D., Schieven G. L., Blake J., Bolen J. B. Cross-linking of surface immunoglobulin activates src-related tyrosine kinases in WEHI 231 cells. Biochem Biophys Res Commun. 1992 Sep 30;187(3):1536–1544. doi: 10.1016/0006-291x(92)90477-3. [DOI] [PubMed] [Google Scholar]
  30. Mantzioris B. X., Berger M. F., Sewell W., Zola H. Expression of the Fc receptor for IgG (Fc gamma RII/CDw32) by human circulating T and B lymphocytes. J Immunol. 1993 Jun 1;150(11):5175–5184. [PubMed] [Google Scholar]
  31. Mitchell M. A., Huang M. M., Chien P., Indik Z. K., Pan X. Q., Schreiber A. D. Substitutions and deletions in the cytoplasmic domain of the phagocytic receptor Fc gamma RIIA: effect on receptor tyrosine phosphorylation and phagocytosis. Blood. 1994 Sep 15;84(6):1753–1759. [PubMed] [Google Scholar]
  32. Muta T., Kurosaki T., Misulovin Z., Sanchez M., Nussenzweig M. C., Ravetch J. V. A 13-amino-acid motif in the cytoplasmic domain of Fc gamma RIIB modulates B-cell receptor signalling. Nature. 1994 Mar 3;368(6466):70–73. doi: 10.1038/368070a0. [DOI] [PubMed] [Google Scholar]
  33. Phillips N. E., Parker D. C. Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis. J Immunol. 1984 Feb;132(2):627–632. [PubMed] [Google Scholar]
  34. Pleiman C. M., Abrams C., Gauen L. T., Bedzyk W., Jongstra J., Shaw A. S., Cambier J. C. Distinct p53/56lyn and p59fyn domains associate with nonphosphorylated and phosphorylated Ig-alpha. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4268–4272. doi: 10.1073/pnas.91.10.4268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pleiman C. M., D'Ambrosio D., Cambier J. C. The B-cell antigen receptor complex: structure and signal transduction. Immunol Today. 1994 Sep;15(9):393–399. doi: 10.1016/0167-5699(94)90267-4. [DOI] [PubMed] [Google Scholar]
  36. Pulford K., Ralfkiaer E., MacDonald S. M., Erber W. N., Falini B., Gatter K. C., Mason D. Y. A new monoclonal antibody (KB61) recognizing a novel antigen which is selectively expressed on a subpopulation of human B lymphocytes. Immunology. 1986 Jan;57(1):71–76. [PMC free article] [PubMed] [Google Scholar]
  37. Reth M. Antigen receptor tail clue. Nature. 1989 Mar 30;338(6214):383–384. doi: 10.1038/338383b0. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Saouaf S. J., Mahajan S., Rowley R. B., Kut S. A., Fargnoli J., Burkhardt A. L., Tsukada S., Witte O. N., Bolen J. B. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9524–9528. doi: 10.1073/pnas.91.20.9524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sarmay G., Rozsnyay Z., Koncz G., Gergely J. Interaction of signaling molecules with human Fc gamma RIIb1 and the role of various Fc gamma RIIb isoforms in B-cell regulation. Immunol Lett. 1995 Jan;44(2-3):125–131. doi: 10.1016/0165-2478(95)00203-h. [DOI] [PubMed] [Google Scholar]
  41. Songyang Z., Carraway K. L., 3rd, Eck M. J., Harrison S. C., Feldman R. A., Mohammadi M., Schlessinger J., Hubbard S. R., Smith D. P., Eng C. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature. 1995 Feb 9;373(6514):536–539. doi: 10.1038/373536a0. [DOI] [PubMed] [Google Scholar]
  42. Songyang Z., Shoelson S. E., Chaudhuri M., Gish G., Pawson T., Haser W. G., King F., Roberts T., Ratnofsky S., Lechleider R. J. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. doi: 10.1016/0092-8674(93)90404-e. [DOI] [PubMed] [Google Scholar]
  43. Stengelin S., Stamenkovic I., Seed B. Isolation of cDNAs for two distinct human Fc receptors by ligand affinity cloning. EMBO J. 1988 Apr;7(4):1053–1059. doi: 10.1002/j.1460-2075.1988.tb02913.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Superti-Furga G. Regulation of the Src protein tyrosine kinase. FEBS Lett. 1995 Aug 1;369(1):62–66. doi: 10.1016/0014-5793(95)00636-n. [DOI] [PubMed] [Google Scholar]
  45. Sármay G., Pecht I., Gergely J. Protein-tyrosine kinase activity tightly associated with human type II Fc gamma receptors. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4140–4144. doi: 10.1073/pnas.91.10.4140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Takata M., Kurosaki T. The catalytic activity of Src-family tyrosine kinase is required for B cell antigen receptor signaling. FEBS Lett. 1995 Nov 6;374(3):407–411. doi: 10.1016/0014-5793(95)01160-g. [DOI] [PubMed] [Google Scholar]
  47. Timson Gauen L. K., Kong A. N., Samelson L. E., Shaw A. S. p59fyn tyrosine kinase associates with multiple T-cell receptor subunits through its unique amino-terminal domain. Mol Cell Biol. 1992 Dec;12(12):5438–5446. doi: 10.1128/mcb.12.12.5438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Van Den Herik-Oudijk I. E., Westerdaal N. A., Henriquez N. V., Capel P. J., Van De Winkel J. G. Functional analysis of human Fc gamma RII (CD32) isoforms expressed in B lymphocytes. J Immunol. 1994 Jan 15;152(2):574–585. [PubMed] [Google Scholar]
  49. Weinrich V., Sondermann P., Bewarder N., Wissel K., Frey J. Epitope mapping of new monoclonal antibodies recognizing distinct human FcRII (CD32) isoforms. Hybridoma. 1996 Apr;15(2):109–116. doi: 10.1089/hyb.1996.15.109. [DOI] [PubMed] [Google Scholar]
  50. Yamanashi Y., Kakiuchi T., Mizuguchi J., Yamamoto T., Toyoshima K. Association of B cell antigen receptor with protein tyrosine kinase Lyn. Science. 1991 Jan 11;251(4990):192–194. doi: 10.1126/science.1702903. [DOI] [PubMed] [Google Scholar]
  51. van de Winkel J. G., Capel P. J. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunol Today. 1993 May;14(5):215–221. doi: 10.1016/0167-5699(93)90166-I. [DOI] [PubMed] [Google Scholar]

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