Temporal Activation of Nontransmembrane Protein-tyrosine Kinases following Mast Cell Fc∊RI Engagement

One of the primary responses observed following antigen-induced cross-linking in mast cells is an increase in the phosphorylation of certain cellular proteins on tyrosine residues. Stimulation of protein-tyrosine kinase activity appears to be necessary for induction of downstream responses such as degranulation. The role of nonreceptor protein-tyrosine kinases in the signal transduction pathway initiated by Fc∊RI engagement in an interleukin-3-dependent mast cell line has been examined. The results presented here show that the enzymatic activity of Lyn is increased within seconds of receptor engagement. Syk activity also undergoes a rapid and transient increase, reaching a peak at approximately 30 s. Similarly, the activity of Fer, representing a third class of nontransmembrane protein-tyrosine kinase increases as well, with its activity peak reached at 1 min poststimulation. The enzymatic activities of Syk and Fer were found to correspond to anti-phosphotyrosine antibody reactivity. Phosphorylation of tyrosine residues of the β and γ chains of Fc∊RI increased concomitant with increased protein-tyrosine kinase activity. These results indicate that at least three classes of nontransmembrane protein-tyrosine kinases are involved in mast cell FceRI signaling and that the activation of these classes of enzymes is temporally regulated. One of the primary responses observed following antigen-induced cross-linking in mast cells is an increase in the phosphorylation of certain cellular proteins on tyrosine residues. Stimulation of protein-tyrosine kinase activity appears to be necessary for induction of downstream responses such as degranulation. The role of nonreceptor protein-tyrosine kinases in the signal transduction pathway initiated by Fc∊RI engagement in an interleukin-3-dependent mast cell line has been examined. The results presented here show that the enzymatic activity of Lyn is increased within seconds of receptor engagement. Syk activity also undergoes a rapid and transient increase, reaching a peak at approximately 30 s. Similarly, the activity of Fer, representing a third class of nontransmembrane protein-tyrosine kinase increases as well, with its activity peak reached at 1 min poststimulation. The enzymatic activities of Syk and Fer were found to correspond to anti-phosphotyrosine antibody reactivity. Phosphorylation of tyrosine residues of the β and γ chains of Fc∊RI increased concomitant with increased protein-tyrosine kinase activity. These results indicate that at least three classes of nontransmembrane protein-tyrosine kinases are involved in mast cell FceRI signaling and that the activation of these classes of enzymes is temporally regulated. INTRODUCTIONMast cells are the primary effectors in immediate type hypersensitivity reactions. Upon exposure to allergen, mast cells are induced to rapidly secrete preformed mediators from cytoplasmic granules. Mast cells bind IgE avidly via specific, high affinity Fc receptors, termed Fc∊RI. Fc∊RI are heterotetrameric complexes comprising an IgE-binding α subunit, a β subunit, and two γ subunits(1Ravetch J.V. Kinet J.-P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1273) Google Scholar, 2Metzger H. Immunol. Rev. 1992; 125: 37-48Crossref PubMed Scopus (222) Google Scholar). The cytoplasmic domains of the β and γ subunits each contain two tyrosine residues, which are located within a conserved consensus sequence termed the immunoreceptor tyrosine activation motif (ITAM) 1The abbreviations used are: ITAMimmunoreceptor tyrosine activation motifMIRRmultichain immune recognition receptorAPTanti-phosphotyrosineDNPdinitrophenolDNP-HSADNP-conjugated human serum albumin. or antigen recognition activation motif(3Samelson L.E. Klausner R.D. J. Biol. Chem. 1992; 267: 24913-24916Abstract Full Text PDF PubMed Google Scholar, 4Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (251) Google Scholar, 5Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar, 6Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1943) Google Scholar). Phosphorylation of this pair of conserved tyrosines is required for successful signal transduction in mast cells (7Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.-P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar) and other hemopoietic cell types(8Sanchez M. Misulovin Z. Burkhardt A.L. Mahajan S. Costa T. Franke R.R. Bolen J.B. Nussenzweig M.C. J. Exp. Med. 1993; 178: 1049-1055Crossref PubMed Scopus (190) Google Scholar, 9Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (876) Google Scholar). Cell activation follows cross-linking of surface-bound IgE molecules by multivalent allergen(10Beaven M.A. Metzger H. Immunol. Today. 1993; 14: 222-226Abstract Full Text PDF PubMed Scopus (365) Google Scholar).Phosphorylation of a select set of cellular proteins on tyrosine residues is a hallmark of the activation of cells through engagement of multichain immune recognition receptors (MIRR) and is one of the earliest detectable events following stimulation of mast cells through Fc∊RI(11Benhamou M. Robbins K.C. Siraganian R.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5327-5330Crossref PubMed Scopus (199) Google Scholar, 12Connelly P.A. Farrell C.A. Merenda J.M. Conklyn M.J. Showell H.J. Biochem. Biophys. Res. Commun. 1991; 177: 192-201Crossref PubMed Scopus (62) Google Scholar, 13Kawakami T. Inagaki N. Takei M. Fukamachi H. Coggeshall K.M. Ishizaka K. Ishizaka T. J. Immunol. 1992; 148: 3513-3519PubMed Google Scholar, 14Yu K.-T. Lyall R. Jariwala N. Zilberstein A. Haimovich J. J. Biol. Chem. 1991; 266: 22564-22568Abstract Full Text PDF PubMed Google Scholar, 15Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (414) Google Scholar). Protein tyrosine phosphorylation following engagement of the Fc∊RI is thought to be mediated by the nontransmembrane protein-tyrosine kinases Lyn and Syk. Lyn, a member of the Src family of protein-tyrosine kinases, is constitutively associated with Fc∊RI(15), binding to the unactivated β subunit(7Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.-P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar). Syk is a 72-kDa protein-tyrosine kinase (16Taniguchi T. Kobayashi T. Kondo J. Takahashi K. Nakamura H. Suzuki J. Nagai K. Yamda T. Nakamura S. Yamaura H. J. Biol. Chem. 1991; 266: 15790-15796Abstract Full Text PDF PubMed Google Scholar) that, along with p70zap (Zap)(9Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (876) Google Scholar), defines a family of cytoplasmic enzymes distinguished by the presence of tandem SH2 domains located amino-terminal to the catalytic domain. Syk becomes activated upon ligation of both B-cell (17Saouaf S.J. Mahajan S. Rowley R.B. Kut S.A. Fargnoli J. Burkhardt A.L. Tsukada S. Witte O.N. Bolen J.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9524-9528Crossref PubMed Scopus (229) Google Scholar) and mast cell MIRRs (7Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.-P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar, 18Minoguchi K. Benhamou M. Swaim W.D. Kawakami Y. Kawakami T. Siraganian R.P. J. Biol. Chem. 1994; 269: 16902-16908Abstract Full Text PDF PubMed Google Scholar) and associates with phosphorylated ITAMs present within the cytoplasmic sequences of the MIRRs. In mast cells, Syk binds to the phosphorylated ITAMs of Fc∊RI γ(19Benhamou M. Ryba N.J.P. Kihara H. Nishikata H. Siraganian R.P. J. Biol. Chem. 1993; 268: 23318-23324Abstract Full Text PDF PubMed Google Scholar, 20Hutchcroft J.E. Geahlen R.L. Deanin G.G. Oliver J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9107-9111Crossref PubMed Scopus (204) Google Scholar, 21Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar, 22Shiue 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. Brugge J.S. Mol. Cell. Biol. 1995; 15: 272-281Crossref PubMed Google Scholar).Fer is a ubiquitously expressed 94-kDa cytoplasmic protein-tyrosine kinase related to Fes(23Hao Q.-L. Heisterkamp N. Groffen J. Mol. Cell. Biol. 1989; 9: 1587-1593Crossref PubMed Scopus (101) Google Scholar). At present, little is known about the involvement of Fer in cellular signaling pathways.Temporal differences in the activation of nonreceptor protein-tyrosine kinases following MIRR engagement have been reported in B-cell and T-cell lines(17Saouaf S.J. Mahajan S. Rowley R.B. Kut S.A. Fargnoli J. Burkhardt A.L. Tsukada S. Witte O.N. Bolen J.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9524-9528Crossref PubMed Scopus (229) Google Scholar, 24Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Prendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar). The purpose of this study was to gain a further understanding of mast cell stimulation by examining the timing and order of activation of nontransmembrane protein-tyrosine kinases following Fc∊RI engagement. We find that Lyn and Syk are activated sequentially and that Fer also undergoes transient activation.MATERIALS AND METHODSAntibodiesRabbit antisera directed against unique sequences of Lyn and Syk have been described previously(24Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Prendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar, 25Burkhardt A.L. Brunswick M. Bolen J.B. Mond J.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3982-3986Crossref PubMed Scopus (354) Google Scholar). Antisera directed against Fc∊RI β amino acids 200-243, Fc∊RI γ amino acids 27-68, and Fer amino acids 201-400 were produced as glutathione S-transferase fusion proteins (Pharmacia Biotech Inc.) and used to immunize rabbits. Mouse monoclonal anti-phosphotyrosine (APT) 5H1 and rabbit polyclonal APT have been described previously(24Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Prendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar, 26Li Z.-H. Mahajan S. Prendergast M. Fargnoli J. Zhu X. Klages S. Adam D. Schieven G.L. Blake J. Bolen J.B. Burkhardt A.L. Biochem. Biophys. Res. Commun. 1992; 187: 1536-1544Crossref PubMed Scopus (35) Google Scholar).CellsPT 18 cells were sensitized by a 1-h preincubation with monoclonal anti-DNP IgE. The cells were washed in serum-free medium and resuspended at a density of 1 × 107 cells/ml. Activation was triggered with the addition of dinitrophenol-conjugated human serum albumin (DNP-HSA).Ca2+ MobilizationThe concentration of intracellular free Ca2+ was measured using the Ca2+ sensitive dye Indo-1 (Molecular Probes, Eugene, OR) and a Coulter EPICS Elite flow cytometer. The procedure used has been described in detail elsewhere(27Rabinovitch P.S. June C.H. Grossmann A. Ledbetter J.A. J. Immunol. 1986; 137: 952-961PubMed Google Scholar). The data were analyzed using Multitime software (Phoenix Flow Systems, San Diego, CA).Immunoprecipitation, Kinase Assays, and ImmunoblottingCell lysis, immunoprecipitation, immune complex kinase assays, and immunoblotting were performed as described previously(28Tsygankov A.Y. Broker B.M. Emmrich F. Cell Immunol. 1992; 140: 97-111Crossref PubMed Scopus (8) Google Scholar, 29Veillette A. Horak I.D. Horak E.M. Bookman M.A. Bolen J.B. Mol. Cell. Biol. 1988; 8: 4353-4361Crossref PubMed Scopus (131) Google Scholar).RESULTSMast Cell Signal Transduction Pathways Are Stimulated by Fc∊RI EngagementPT 18 mouse mast cells were sensitized by incubating the cells for 1 h with an allergen-specific IgE (anti-DNP). The sensitized cells were then stimulated by the addition of DNP-HSA and lysed as a function of time over the course of 30 min. The lysates were analyzed by anti-phosphotyrosine immunoblotting to establish the extent and timing of protein tyrosine phosphorylation a complex of protein tyrosine phosphorylation and with Fc∊RI engagement results in the immediate tyrosine phosphorylation of a of with the at of and In the of tyrosine phosphorylation a peak at and has to at of cells to DNP-HSA was also by in the concentration of intracellular free The for DNP-HSA is in that addition of of increases in a increase in that is and a that the The of 18 cells to to high of DNP-HSA are in Phosphorylation of Fc∊RI the proteins phosphorylated on tyrosine following receptor cross-linking are the β and γ subunits of Fc∊RI. the of receptor the tyrosine phosphorylation of β and γ was as a function of time receptor engagement phosphorylation of and be as as following Fc∊RI engagement. Phosphorylation of both β and γ follows the The peak of tyrosine phosphorylation at 1 min and to by 30 min poststimulation. The of and was over the time that in and APT increased tyrosine upon a of of has an of approximately with an of for the the of by with the APT is that the phosphorylated that appears activation for a of the in the course of and tyrosine cells were stimulated with lysed at the and with for the phosphorylation was by APT immunoblotting and was by or immunoblotting of phosphorylated and protein are of Lyn, and cells were stimulated by with and lysates were over a time course as described The enzymatic activity of protein-tyrosine kinases in these was through the of in kinase The kinase activity of Lyn increased following receptor reaching its at the time of s. a of Lyn activation is with activity to an by 30 The Src family member is also produced by cells at a detectable course of Lyn, and Fer cells were stimulated with lysed at the and with for the protein-tyrosine The protein kinase activities of the protein-tyrosine kinases were in immune complex or immune complex kinase using rabbit as an catalytic activity of Syk was also found to increase following Fc∊RI engagement. Syk activity was its peak of activity about 30 The activity then to by 30 kinase activity of Fer was also to Fer be for the protein at approximately by APT The protein kinase activity of Fer was found to increase following addition of Fer activity was increased by with a by 1 min. Fer activity was to by activity of Lyn, and Fer by were by the of these enzymes to an In each the of each to the phosphorylation of in in the Lyn was activated the of the and stimulated activities is observed in the The of Syk to with its to Similarly, the kinase activity of Fer that observed in the the enzymatic activation and tyrosine phosphorylation of these enzymes was by of each protein with APT following Fc∊RI engagement. The results of these are presented in Syk was to APT antibody from cells, its increased following receptor engagement APT was high from 30 to and then to a detectable by 30 min. The of Syk was in the course of these the of Syk with APT antibody its enzymatic activation as by both and phosphorylation of The APT of Fer also increased upon stimulation of the cells, reaching a at 30 to 1 min and to by 30 min. The of Fer was over this time observed with in the tyrosine phosphorylation of Fer with in its enzymatic course of tyrosine phosphorylation of Syk and Fer cells were stimulated with lysed at the and with for the protein-tyrosine phosphorylation was by APT immunoblotting and was by kinase immunoblotting of Fer, and protein are tyrosine phosphorylation is a of signal transduction triggered in to receptor engagement in hemopoietic The of an activation of enzymes necessary to such has been in both and S.J. Mahajan S. Rowley R.B. Kut S.A. Fargnoli J. Burkhardt A.L. Tsukada S. Witte O.N. Bolen J.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9524-9528Crossref PubMed Scopus (229) Google Scholar, 24Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Prendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar). We have found that this to mast cells, with a time that in addition to Lyn and the nonreceptor protein-tyrosine kinase Fer in mast cell signaling and is of the activation results of immune complex kinase show that the enzymatic activation of the Src family member Lyn Fc∊RI cross-linking is immediate and The activation of Src family enzymes in MIRR signal transduction is a and are activated in to T-cell receptor A.L. Stealey B. Rowley R.B. Mahajan S. Prendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar), the enzymatic activities of Lyn and are increased following stimulation of B-cell through the B-cell S.J. Mahajan S. Rowley R.B. Kut S.A. Fargnoli J. Burkhardt A.L. Tsukada S. Witte O.N. Bolen J.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9524-9528Crossref PubMed Scopus (229) Google Scholar), and Lyn has been to be activated in stimulated mast cell and cell M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.-P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar, 15Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (414) Google Scholar). Lyn is the Src family member in 18 cells and other mast cell and cell and appears to the signal following Fc∊RI engagement. The of Lyn at the cytoplasmic of the and the that this protein-tyrosine kinase is constitutively to the this in the to rapidly to receptor peak of Syk and Fer activation were found to that of The peak of Syk activity with the time at which tyrosine phosphorylation is is with a Lyn is activated by receptor cross-linking and the β and γ subunits of the γ ITAMs a for Syk via its tandem SH2 H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar, 22Shiue 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. Brugge J.S. Mol. Cell. Biol. 1995; 15: 272-281Crossref PubMed Google Scholar). that binding of Syk to phosphorylated ITAMs a in the to its enzymatic R.B. Burkhardt A.L. Bolen J.B. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google timing of the and the of Syk activation in mast cells are from observed in S.J. Mahajan S. Rowley R.B. Kut S.A. Fargnoli J. Burkhardt A.L. Tsukada S. Witte O.N. Bolen J.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9524-9528Crossref PubMed Scopus (229) Google Scholar). present a complex of proteins following engagement of the B-cell antigen and the tyrosine phosphorylation this is in In cells for tyrosine phosphorylation of α and activation and tyrosine phosphorylation of Syk are both for 1 The in 18 mast cells is by a time the cell tyrosine phosphorylation as as the activation of protein-tyrosine kinases to of activity by 30 min poststimulation. time is with the role of the mast cell to an immediate hypersensitivity and then protein-tyrosine kinase Fer is also activated following engagement of the Fc∊RI. The catalytic activation and tyrosine phosphorylation of Fer follows that of Syk. is the known of Fer activation in to are performed to the role of Fer in cell signal protein-tyrosine kinase reported to a role in mast cell signal transduction is L. Kawakami Y. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). found to be an in cells, have been to activation or an increase in tyrosine phosphorylation of following stimulation of cells The present in 18 cells appears to have a high of tyrosine high of tyrosine phosphorylation in the of stimulation in the activation of following receptor cross-linking or to such have that tyrosine phosphorylation in 18 mast cells is rapid and classes of protein-tyrosine kinases are activated in a following Fc∊RI engagement. The Src family member Lyn is activated by activation of Syk and These that Lyn is the of the protein-tyrosine kinase that results in mast cell degranulation. INTRODUCTIONMast cells are the primary effectors in immediate type hypersensitivity reactions. Upon exposure to allergen, mast cells are induced to rapidly secrete preformed mediators from cytoplasmic granules. Mast cells bind IgE avidly via specific, high affinity Fc receptors, termed Fc∊RI. Fc∊RI are heterotetrameric complexes comprising an IgE-binding α subunit, a β subunit, and two γ subunits(1Ravetch J.V. Kinet J.-P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1273) Google Scholar, 2Metzger H. Immunol. Rev. 1992; 125: 37-48Crossref PubMed Scopus (222) Google Scholar). The cytoplasmic domains of the β and γ subunits each contain two tyrosine residues, which are located within a conserved consensus sequence termed the immunoreceptor tyrosine activation motif (ITAM) 1The abbreviations used are: ITAMimmunoreceptor tyrosine activation motifMIRRmultichain immune recognition receptorAPTanti-phosphotyrosineDNPdinitrophenolDNP-HSADNP-conjugated human serum albumin. or antigen recognition activation motif(3Samelson L.E. Klausner R.D. J. Biol. Chem. 1992; 267: 24913-24916Abstract Full Text PDF PubMed Google Scholar, 4Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (251) Google Scholar, 5Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar, 6Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1943) Google Scholar). Phosphorylation of this pair of conserved tyrosines is required for successful signal transduction in mast cells (7Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.-P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar) and other hemopoietic cell types(8Sanchez M. Misulovin Z. Burkhardt A.L. Mahajan S. Costa T. Franke R.R. Bolen J.B. Nussenzweig M.C. J. Exp. Med. 1993; 178: 1049-1055Crossref PubMed Scopus (190) Google Scholar, 9Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (876) Google Scholar). Cell activation follows cross-linking of surface-bound IgE molecules by multivalent allergen(10Beaven M.A. Metzger H. Immunol. Today. 1993; 14: 222-226Abstract Full Text PDF PubMed Scopus (365) Google Scholar).Phosphorylation of a select set of cellular proteins on tyrosine residues is a hallmark of the activation of cells through engagement of multichain immune recognition receptors (MIRR) and is one of the earliest detectable events following stimulation of mast cells through Fc∊RI(11Benhamou M. Robbins K.C. Siraganian R.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5327-5330Crossref PubMed Scopus (199) Google Scholar, 12Connelly P.A. Farrell C.A. Merenda J.M. Conklyn M.J. Showell H.J. Biochem. Biophys. Res. Commun. 1991; 177: 192-201Crossref PubMed Scopus (62) Google Scholar, 13Kawakami T. Inagaki N. Takei M. Fukamachi H. Coggeshall K.M. Ishizaka K. Ishizaka T. J. Immunol. 1992; 148: 3513-3519PubMed Google Scholar, 14Yu K.-T. Lyall R. Jariwala N. Zilberstein A. Haimovich J. J. Biol. Chem. 1991; 266: 22564-22568Abstract Full Text PDF PubMed Google Scholar, 15Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (414) Google Scholar). Protein tyrosine phosphorylation following engagement of the Fc∊RI is thought to be mediated by the nontransmembrane protein-tyrosine kinases Lyn and Syk. Lyn, a member of the Src family of protein-tyrosine kinases, is constitutively associated with Fc∊RI(15), binding to the unactivated β subunit(7Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.-P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar). Syk is a 72-kDa protein-tyrosine kinase (16Taniguchi T. Kobayashi T. Kondo J. Takahashi K. Nakamura H. Suzuki J. Nagai K. Yamda T. Nakamura S. Yamaura H. J. Biol. Chem. 1991; 266: 15790-15796Abstract Full Text PDF PubMed Google Scholar) that, along with p70zap (Zap)(9Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (876) Google Scholar), defines a family of cytoplasmic enzymes distinguished by the presence of tandem SH2 domains located amino-terminal to the catalytic domain. Syk becomes activated upon ligation of both B-cell (17Saouaf S.J. Mahajan S. Rowley R.B. Kut S.A. Fargnoli J. Burkhardt A.L. Tsukada S. Witte O.N. Bolen J.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9524-9528Crossref PubMed Scopus (229) Google Scholar) and mast cell MIRRs (7Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.-P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar, 18Minoguchi K. Benhamou M. Swaim W.D. Kawakami Y. Kawakami T. Siraganian R.P. J. Biol. Chem. 1994; 269: 16902-16908Abstract Full Text PDF PubMed Google Scholar) and associates with phosphorylated ITAMs present within the cytoplasmic sequences of the MIRRs. In mast cells, Syk binds to the phosphorylated ITAMs of Fc∊RI γ(19Benhamou M. Ryba N.J.P. Kihara H. Nishikata H. Siraganian R.P. J. Biol. Chem. 1993; 268: 23318-23324Abstract Full Text PDF PubMed Google Scholar, 20Hutchcroft J.E. Geahlen R.L. Deanin G.G. Oliver J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9107-9111Crossref PubMed Scopus (204) Google Scholar, 21Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar, 22Shiue 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. Brugge J.S. Mol. Cell. Biol. 1995; 15: 272-281Crossref PubMed Google Scholar).Fer is a ubiquitously expressed 94-kDa cytoplasmic protein-tyrosine kinase related to Fes(23Hao Q.-L. Heisterkamp N. Groffen J. Mol. Cell. Biol. 1989; 9: 1587-1593Crossref PubMed Scopus (101) Google Scholar). At present, little is known about the involvement of Fer in cellular signaling pathways.Temporal differences in the activation of nonreceptor protein-tyrosine kinases following MIRR engagement have been reported in B-cell and T-cell lines(17Saouaf S.J. Mahajan S. Rowley R.B. Kut S.A. Fargnoli J. Burkhardt A.L. Tsukada S. Witte O.N. Bolen J.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9524-9528Crossref PubMed Scopus (229) Google Scholar, 24Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Prendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar). The purpose of this study was to gain a further understanding of mast cell stimulation by examining the timing and order of activation of nontransmembrane protein-tyrosine kinases following Fc∊RI engagement. We find that Lyn and Syk are activated sequentially and that Fer also undergoes transient