Adaptor Protein 3BP2 Is a Potential Ligand of Src Homology 2 and 3 Domains of Lyn Protein-tyrosine Kinase

Abstract

Adaptor protein 3BP2, a c-Abl-Src homology 3 (SH3) domain-binding protein, is known to play a regulatory role in T-cell receptor-mediated transcriptional activation of nuclear factor of activated T cell and activator protein 1 by interacting with Syk/ZAP-70 protein-tyrosine kinase. We have previously demonstrated that aggregation of high affinity IgE receptor (FcϵRI) induces tyrosine phosphorylation of 3BP2, and overexpression of the 3BP2-SH2 domain suppresses antigen-induced degranulation in rat basophilic leukemia RBL-2H3 mast cell line. In this report, we attempt to analyze the biological relevance of 3BP2 tyrosine phosphorylation. By using the transient expression system in COS-7 cells, we have demonstrated that 3BP2 was predominantly phosphorylated on Tyr174, Tyr183, and Tyr446 when it was coexpressed with Syk. An in vitro binding study revealed that phosphorylation of Tyr446 by Syk was likely to create a binding site for the Lyn-SH2 domain in RBL-2H3 cells. In addition, proline-rich region of 3BP2 bound to the Lyn-SH3 domain. Conformational microscopic analysis showed that Lyn and 3BP2 are constitutively colocalized in RBL-2H3 cells. Overexpression of 3BP2 in RBL-2H3 cells resulted in an enhancement of Lyn autophosphorylation. These results suggest that the adaptor protein 3BP2 is a potential regulator of Lyn protein-tyrosine kinase as a ligand of its SH3/SH2 domains in FcϵRI-mediated signaling in mast cells. Adaptor protein 3BP2, a c-Abl-Src homology 3 (SH3) domain-binding protein, is known to play a regulatory role in T-cell receptor-mediated transcriptional activation of nuclear factor of activated T cell and activator protein 1 by interacting with Syk/ZAP-70 protein-tyrosine kinase. We have previously demonstrated that aggregation of high affinity IgE receptor (FcϵRI) induces tyrosine phosphorylation of 3BP2, and overexpression of the 3BP2-SH2 domain suppresses antigen-induced degranulation in rat basophilic leukemia RBL-2H3 mast cell line. In this report, we attempt to analyze the biological relevance of 3BP2 tyrosine phosphorylation. By using the transient expression system in COS-7 cells, we have demonstrated that 3BP2 was predominantly phosphorylated on Tyr174, Tyr183, and Tyr446 when it was coexpressed with Syk. An in vitro binding study revealed that phosphorylation of Tyr446 by Syk was likely to create a binding site for the Lyn-SH2 domain in RBL-2H3 cells. In addition, proline-rich region of 3BP2 bound to the Lyn-SH3 domain. Conformational microscopic analysis showed that Lyn and 3BP2 are constitutively colocalized in RBL-2H3 cells. Overexpression of 3BP2 in RBL-2H3 cells resulted in an enhancement of Lyn autophosphorylation. These results suggest that the adaptor protein 3BP2 is a potential regulator of Lyn protein-tyrosine kinase as a ligand of its SH3/SH2 domains in FcϵRI-mediated signaling in mast cells. Adaptor protein 3BP2 was originally isolated as a protein-tyrosine kinase c-Abl-Src homology 3 (SH3) 1The abbreviations used are: SH, Src homology; FcϵRI, high affinity IgE receptor; PTK, protein-tyrosine kinase; ITAM, immunoreceptor tyrosine-based activating motif; GST, glutathione S-transferase; HA, hemagglutinin epitope; IPTG, isopropyl-β-d-thiogalactopyranoside; DNP, dinitrophenyl; mAb, monoclonal antibody; BSA, bovine serum albumin; WT, wild type; PBS, phosphate-buffered saline. domain-binding protein of unknown function (1Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Google Scholar). 3BP2 was also identified as a Syk kinase-interacting protein by the yeast two-hybrid screening (2Deckert M. Tartare-Deckert S. Hernandez J. Rottapel R. Altman A. Immunity. 1998; 9: 595-605Google Scholar). Transient overexpression of 3BP2 resulted in a transcriptional activation of nuclear factor of activated T cell and activator protein 1, which is induced by T-cell receptor aggregation. Ser225 and Ser277 of 3BP2 were identified as essential sites for interacting with 14-3-3 to negatively regulate the function of 3BP2 in lymphocytes (3Foucault I. Liu Y.C. Bernard A. Deckert M. J. Biol. Chem. 2003; 278: 7146-7153Google Scholar). Moreover, infection of 3BP2 wild type into NK cells by vaccinia virus enhanced cell cytotoxicity. An in vitro binding study suggested that phosphorylation of Tyr183 of 3BP2 could be associated with Vav and phospholipase C-γ (4Jevremovic D. Billadeau D.D. Schoon R.A. Dick C.J. Leibson P.J. J. Immunol. 2001; 166: 7219-7228Google Scholar). In mast cells, overexpression of the 3BP2-SH2 domain suppressed high affinity IgE receptor (FcϵRI)-mediated tyrosine phosphorylation of phospholipase C-γ, Ca2+ mobilization, and degranulation (5Sada K. Miah S.M.S. Maeno K. Kyo S. Qu X. Yamamura H. Blood. 2002; 100: 2138-2144Google Scholar). These findings have demonstrated that 3BP2 plays a critical role in hematopoietic cells. To propagate the immunoreceptor signal, adaptor proteins contribute to protein-protein and protein-lipid interactions through multiple domains and/or specific phosphotyrosine-containing sequences. Tyrosine phosphorylation of 3BP2 was observed in NK cells and mast cells by cross-linking FcγR and FcϵRI, respectively (4Jevremovic D. Billadeau D.D. Schoon R.A. Dick C.J. Leibson P.J. J. Immunol. 2001; 166: 7219-7228Google Scholar, 5Sada K. Miah S.M.S. Maeno K. Kyo S. Qu X. Yamamura H. Blood. 2002; 100: 2138-2144Google Scholar). To elucidate the function of 3BP2, it is necessary to determine the protein-tyrosine kinase (PTK) that phosphorylates 3BP2 and its binding partner to assemble a signaling complex through specific phosphotyrosine-containing motifs in 3BP2. The Src family PTK Lyn is associated with FcϵRIβ. Upon aggregation of FcϵRI, Lyn is critical for phosphorylating FcϵRIβ and -γ subunits on Tyr residues within the immunoreceptor tyrosine-based activating motif (ITAM) (6Paolini R. Jouvin M.H. Kinet J.P. Nature. 1991; 353: 855-858Google Scholar, 7Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Google Scholar, 8Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.P. J. Biol. Chem. 1994; 269: 5918-5925Google Scholar). By analogy with studies on Hck, Lyn is thought to be activated by the disassembly of the closed intramolecular interaction by (i) CD45-mediated dephosphorylation of C-terminal regulatory Tyr residue, (ii) binding to SH3 and SH2 ligands, and (iii) autophosphorylation of Tyr in the activation loop (9Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Google Scholar). What is the binding ligand of the SH3 and SH2 domains of Lyn in FcϵRI signaling pathway? Pull-down experiments using glutathione S-transferase (GST)-Lyn-SH2 fusion protein indicated that there were multiple phosphoproteins interacting with the Lyn-SH2 after the antigen stimulation of RBL-2H3 cells (10Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Google Scholar). In addition, although the displacement of intramolecular SH3 interaction is not well understood, it seems likely that some aggregation-induced change in an associated molecule provides a higher affinity SH3 ligand that binds to the Lyn-SH3 domain (11Turner H. Kinet J.P. Nature. 1999; 402: B24-B30Google Scholar). The SH3 domain is directed toward the proline-rich region, but such a ligand has not been identified yet in the FcϵRI signaling. We isolated nonreceptor type PTK Syk from porcine spleen (12Taniguchi T. Kobayashi T. Kondo J. Takahashi K. Nakamura H. Suzuki J. Nagai K. Yamada T. Nakamura S. Yamamura H. J. Biol. Chem. 1991; 266: 15790-15796Google Scholar). Syk is expressed in hematopoietic, epithelial, and endothelial cells (13Turner M. Schweighoffer E. Colucci F. Di Santo J.P. Tybulewicz V.L. Immunol. Today. 2000; 21: 148-154Google Scholar, 14Sada K. Takano T. Yanagi S. Yamamura H. J. Biochem. (Tokyo). 2001; 130: 177-186Google Scholar, 15Coopman P.J. Do M.T. Barth M. Bowden E.T. Hayes A.J. Basyuk E. Blancato J.K. Vezza P.R. McLeskey S.W. Mangeat P.H. Mueller S.C. Nature. 2000; 406: 742-747Google Scholar, 16Yanagi S. Inatome R. Ding J. Kitaguchi H. Tybulewicz V.L. Yamamura H. Blood. 2001; 98: 2869-2871Google Scholar). When the ITAM of FcϵRIγ subunits is phosphorylated by Lyn, Syk is recruited to the plasma membrane by binding its tandem SH2 domain and is autophosphorylated (17Siraganian R.P. Zhang J. Suzuki K. Sada K. Mol. Immunol. 2002; 38: 1229-1233Google Scholar). Syk has multiple autophosphorylation sites. Tyr317, Tyr342, and Tyr346 are located in the linker region of Syk; Tyr519 and Tyr520 are in the activation loop of the kinase domain; and Tyr624 and Tyr625 are in the C-terminal region (18Furlong M.T. Mahrenholz A.M. Kim K.H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Google Scholar). Phosphorylation of Tyr519 and Tyr520 is critical for the enzymatic activation of Syk (19Zhang J. Billingsley M.L. Kincaid R.L. Siraganian R.P. J. Biol. Chem. 2000; 275: 35442-35447Google Scholar). Another member of the Syk family PTKs, ZAP-70, has a similar structural feature. However, there are differences in the binding molecules and mechanism of enzymatic activation between Syk and ZAP-70 (13Turner M. Schweighoffer E. Colucci F. Di Santo J.P. Tybulewicz V.L. Immunol. Today. 2000; 21: 148-154Google Scholar, 14Sada K. Takano T. Yanagi S. Yamamura H. J. Biochem. (Tokyo). 2001; 130: 177-186Google Scholar). In the present study, we provide evidence that Tyr174, Tyr183, and Tyr446 on 3BP2 are phosphorylated by a nonreceptor type PTK, Syk. Phosphorylation of 3BP2 on Tyr446 creates a binding site for the Lyn-SH2 domain in vitro. Additional interaction between the proline-rich region and Lyn-SH3 may contribute to the constitutive codistribution of both molecules in RBL-2H3 cells. Furthermore, overexpression of 3BP2 resulted in an enhancement of antigen-induced Lyn autophosphorylation in RBL-2H3 cells. On the other hand, overexpression of 3BP2-SH2 resulted in a suppression of Lyn autophosphorylation. Our results suggest that 3BP2 is a potential ligand of Lyn-SH3/SH2 domains that positively regulates the autophosphorylation of Lyn in mast cells. Here, we are proposing the model of a novel Lyn-activating cycle in mast cells. Reagents and Antibodies—Protein A-agarose and mouse monoclonal anti-dinitrophenyl IgE (anti-DNP IgE, clone SPE-7) were purchased from Sigma. was from and were from was from was from Syk was as previously K. Yamamura H. J. Biochem. 1997; Scholar). were from bovine serum of 1 of was from Suzuki T. M. T. S. M. K. J. Biol. Chem. 1997; Scholar). of expression of mouse 3BP2 in was by A. Altman of Tyr174, Tyr183, and Tyr446 of to were by the The of function of the 3BP2-SH2 domain was previously (5Sada K. Miah S.M.S. Maeno K. Kyo S. Qu X. Yamamura H. Blood. 2002; 100: 2138-2144Google Scholar). of proline-rich region was by The wild type and of 3BP2 used in this study were in 1, and were by of porcine Syk and SH2 domain that SH2 domains and were previously K. Yamamura H. J. Biochem. 1997; Scholar). The Lyn, kinase from R. P. of and from T. were into expression was from T. and Syk was from and into expression of and basophilic leukemia RBL-2H3 cells and COS-7 cells were as in with of and wild type was into RBL-2H3 cells by the expression system of was into cells by using to of were by the of protein expression by of cell with The cells the of were used as cells for the of in the were into cells by were with and of were by the of expression of using the of cell with an cell were also by with mAb, by R. P. were for The were with 1 for to cell The RBL-2H3 cells were previously (5Sada K. Miah S.M.S. Maeno K. Kyo S. Qu X. Yamamura H. Blood. 2002; 100: 2138-2144Google Scholar). cell RBL-2H3 cells were with IgE were with 1 and and with antigen in the for the indicated transient of COS-7 cells, 1 of and of were to cells in a to the after cells were used for the of and cells were with and in 1 1 and were by the and were with the indicated to protein A-agarose for 1 the were with the proteins were by for with of the of were used as cell were by and The were with in and for 1 were with and with the indicated in for 1 in the were with for and in In proteins were by the enhanced Pull-down Lyn-SH2 domain expression was by P. Siraganian (10Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Google Scholar). The Lyn-SH3 domain was by using the and sites are The was into the to a domain with The cells by were in and the cell were by the were with for 1 The were with and with the binding 1 and RBL-2H3 COS-7 cells with the of were in the binding The cell were with the indicated fusion protein to for 1 The were with the binding and proteins were by for with In were in of the kinase of for were by for with and proteins were by The was with 1 for 1 to and of proteins were by using Lyn were by the with RBL-2H3 cells were a in and with 1 a cells were with IgE and for of with were with PBS, with in for and with in for cells were for in and with in cells were with and for 1 with in PBS, and with the and for the cells were using and by using system Inatome R. Takahashi S. Yamamura H. Yanagi S. J. 2002; 21: Scholar). Tyrosine Syk Tyr174, Tyr183, and Tyr446 in previously the FcϵRI-mediated tyrosine phosphorylation of the adaptor protein 3BP2 in RBL-2H3 mast cells (5Sada K. Miah S.M.S. Maeno K. Kyo S. Qu X. Yamamura H. Blood. 2002; 100: 2138-2144Google Scholar). 3BP2 is and a 1 after that nonreceptor type may contribute to tyrosine phosphorylation of 3BP2. In COS-7 cells, 3BP2 was by Lyn, known to be activated after FcϵRI aggregation in mast cells In 3BP2 was predominantly phosphorylated by which is known to play an essential role in and receptor-mediated activation of hematopoietic cells (13Turner M. Schweighoffer E. Colucci F. Di Santo J.P. Tybulewicz V.L. Immunol. Today. 2000; 21: 148-154Google Scholar, 14Sada K. Takano T. Yanagi S. Yamamura H. J. Biochem. (Tokyo). 2001; 130: 177-186Google Scholar). on this we to the tyrosine residues in 3BP2 phosphorylated by Syk. the study showed that Syk the in its R. H. J. Mol. Biol. Scholar). 3BP2 has and located in the In addition, an in vitro binding study suggested that phosphorylation of Tyr183 located in motif creates a binding site for the domain (4Jevremovic D. Billadeau D.D. Schoon R.A. Dick C.J. Leibson P.J. J. Immunol. 2001; 166: 7219-7228Google Scholar). we a of 3BP2 in which Tyr was to of Tyr residues to phosphorylation in COS-7 cells, that some of residues are the phosphorylation sites by Syk 3 and with the 3BP2 in which Tyr residues were for and were by Syk in COS-7 cells that Syk phosphorylates Tyr residues in 3BP2. The of 3BP2 with Lyn resulted in a tyrosine phosphorylation of 3BP2 to Lyn could Tyr174, Tyr183, and Tyr446 in COS-7 cells not of the and 3BP2 were by the 1, and These results that Tyr174, Tyr183, and Tyr446 in 3BP2 are the tyrosine phosphorylation sites by nonreceptor type PTKs, in Syk. a to the Lyn-SH2 the role of 3BP2 tyrosine we the RBL-2H3 cells in which the expression of is induced by the of we aggregation of FcϵRI induced a tyrosine phosphorylation of 3BP2 in RBL-2H3 cells (5Sada K. Miah S.M.S. Maeno K. Kyo S. Qu X. Yamamura H. Blood. 2002; 100: 2138-2144Google Scholar). Phosphorylation of 3BP2 was after antigen stimulation and to To the binding of 3BP2, experiments were by using fusion proteins mast cells not with from cell from cells, that antigen-induced tyrosine phosphorylation of 3BP2 creates the binding site for Lyn-SH2 in mast cells To the mechanism of this a system using COS-7 cells was by transient of but not was bound to Lyn-SH2 when it was coexpressed with Syk Phosphorylation of 3BP2 by Lyn could not interaction with Lyn-SH2 in COS-7 cells, to the of 3BP2 tyrosine phosphorylation as in on we to the Tyr that associated with Lyn-SH2 by using this The and 3BP2 in which Tyr residues were for and was with and cell was with Tyr the binding of for Lyn-SH2 was similar to that of wild type a binding with and not with Lyn-SH2 and that phosphorylation of Tyr446 by Syk creates binding site for was 3BP2 in which Tyr was for was with and cell was with with the expression of 3BP2 with Syk resulted in a interaction with Lyn-SH2 of Tyr183 into not binding with Lyn-SH2 and In the experiments using COS-7 cells, the expression of PTK and the of 3BP2 were by the of cell results suggest that antigen stimulation induces tyrosine phosphorylation of 3BP2, the interaction with Moreover, tyrosine phosphorylation sites in 3BP2, phosphorylation of Tyr446 to be the site to the interaction with Lyn-SH2 FcϵRI aggregation in mast cells. Phosphorylation of Tyr446 but the SH2 of 3BP2 to the of Lyn with binds to Syk in RBL-2H3 mast cells (10Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Google Scholar). demonstrated that Syk was with when it was coexpressed with in COS-7 cells of Syk was with Lyn-SH2 a of Tyr446 of 3BP2 the that phosphorylation of Tyr446 of 3BP2 by Syk is for the binding of Syk with Lyn-SH2 3BP2 is necessary for Syk to with Lyn-SH2 in COS-7 cells, Syk could not the Tyr to with in mast cells (10Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Google Scholar). a of into in 3BP2-SH2 on the with although 3BP2-SH2 was to with Syk in yeast (2Deckert M. Tartare-Deckert S. Hernandez J. Rottapel R. Altman A. Immunity. 1998; 9: 595-605Google Scholar). of the SH2 domain of Syk resulted in an in the kinase and tyrosine phosphorylation of Syk S. T. Yamamura H. Scholar). the wild the SH2 of Syk was of with of 3BP2 tyrosine phosphorylation 1 and The multiple of to be to its autophosphorylation sites Tyr342, and 1 and (18Furlong M.T. Mahrenholz A.M. Kim K.H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Google Scholar, R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; Scholar). that Lyn-SH2 could to the autophosphorylation site of in COS-7 cells. To with wild type Syk Syk with multiple phosphorylation not 3BP2. The expression of 3BP2, and were by the of cell and results suggest that 3BP2 tyrosine phosphorylation of Phosphorylation of Tyr446 is critical for this function of 3BP2. of 3BP2 with Lyn-SH3 was isolated as a domain-binding protein its proline-rich region (1Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Google Scholar). 3BP2 was of binding to SH3 domain of (1Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Google Scholar, M. Tartare-Deckert S. Hernandez J. Rottapel R. Altman A. Immunity. 1998; 9: 595-605Google Scholar). we have demonstrated that 3BP2 binds to the Lyn-SH2 we the SH3 domain of Lyn could with 3BP2. The binding of 3BP2 with the Lyn-SH3 domain was observed when domain with cell from both and mast cells The expression of was by the of cell To the mechanism of this a system using COS-7 cells was the Lyn-SH2 bound to 3BP2 when it was coexpressed with Lyn-SH3 could to 3BP2 phosphorylation by Syk of Tyr446 not the interaction of both The interaction between 3BP2 and Lyn-SH3 was by the of the proline-rich region in 3BP2 3BP2 with the of the proline-rich region and a was bound to this indicated that proline-rich region could with Lyn-SH3 domain. The expression of Syk and the of 3BP2 were by the of cell and both Lyn-SH2 and SH3 domains bound to the 3BP2 The binding of the 3BP2 with Lyn-SH2 was observed when the cells were with the the interaction with Lyn-SH3 domain was also both in and cells. analysis by revealed that 3BP2 and Lyn were colocalized in the plasma membrane in and RBL-2H3 cells 3BP2 was not in cells, the interaction of the 3BP2 proline-rich region with Lyn-SH3 may contribute to the of both molecules in mast cells. Overexpression of of we to the role of the interaction of 3BP2 with with an overexpression of 3BP2 wild type were with an and cell were with were to an in vitro protein kinase Overexpression of 3BP2 resulted in an in the autophosphorylation of Lyn antigen stimulation in RBL-2H3 cells The analysis revealed that expression of 3BP2 resulted in a in the kinase of Lyn after antigen stimulation with that of cells, the was in cells Furthermore, overexpression of the of 3BP2, resulted in the in the autophosphorylation of Lyn of and was by the of cell and results indicated that overexpression of the autophosphorylation of Lyn to in its kinase of Lyn with the 3BP2 positively regulates the kinase of We previously demonstrated that the SH2 domain of 3BP2 positively regulates FcϵRI-mediated tyrosine phosphorylation of phospholipase C-γ, mobilization, and degranulation in RBL-2H3 cells (5Sada K. Miah S.M.S. Maeno K. Kyo S. Qu X. Yamamura H. Blood. 2002; 100: 2138-2144Google Scholar). In this report, we have that the proline-rich region and phosphorylated Tyr446 of 3BP2 bound to Lyn-SH3 and respectively and that 3BP2 is a ligand of Lyn in FcϵRI signaling In cells, a proline-rich region may 3BP2 to the and the receptor aggregation creates the high affinity site by phosphorylating a homology domain with in to the of 3BP2 in cells. In COS-7 cells, the expression of 3BP2 enhanced Syk tyrosine phosphorylation 1 and is that 3BP2 the Lyn to Tyr346 in the linker region of which could to the Lyn-SH2 in COS-7 cells phosphorylation site in ZAP-70 is a binding site for the SH2 domain of a member of Src family M. Di D. E. A. O. J. Biol. Chem. 1999; Scholar, B.J. E. M. J. 1999; Scholar). However, findings revealed that phosphorylation of Tyr346 is for FcϵRI-mediated mast cell activation J. E. Siraganian R.P. Mol. Cell. Biol. 2002; Scholar). Another is that 3BP2 Lyn to the protein, which and Syk. Phosphorylation of Tyr519 and Tyr520 in its activation loop of Syk creates the binding sites to the SH2 domain of Deckert M. S. Altman A. T. J. Biol. Chem. Scholar). suggest that 3BP2 is a ligand of Lyn-SH3/SH2 domains enzymatic activation of Lyn in RBL-2H3 cells An adaptor protein is thought to function to signaling molecules by interacting with signaling domains and specific motifs Our present study that an adaptor molecule has a role to enzymatic activation of PTKs, by of FcϵRI induces activation of Lyn, which phosphorylates tyrosine in the ITAM of FcϵRIβ and -γ subunits to and Syk. studies have demonstrated that an expression of Lyn is necessary to Syk to in M. H. A. T. T. Yamamura H. T. J. 1994; Scholar). These that Lyn is of Syk J.P. Immunol. 1999; Scholar). is that 3BP2 is the of Syk. When 3BP2 is by it of the FcϵRI-mediated signaling by the kinase of Lyn 3BP2 may function to the antigen-induced mast there is an evidence of the activation mechanism of Syk Syk is associated with the T-cell receptor to the and induces activation of of Altman A. T. S. A. 1994; Scholar, H. Bolen J.B. A. J. Scholar). In addition, Syk tyrosine phosphorylation of to the kinase of Syk by S. M. A. Mol. Cell. Biol. 1997; Scholar). We have demonstrated that 3BP2 is by Syk in COS-7 cells. Phosphorylation of Tyr446 of 3BP2 by Syk as a to Lyn of Syk in the could have a regulatory role for Lyn, receptor aggregation may the of the Syk T. Immunol. 1999; Scholar). findings the that Lyn could be by Syk in FcϵRI signaling in mast cells 3BP2 may contribute the activation mechanism of Lyn in mast cells. The kinase of Src family has been thought to be by C-terminal Src kinase and in hematopoietic cells. The present study the of the model of Lyn activation cycle in study by Yanagi S. H. M. H. Yamamura H. T. J. Biol. Chem. demonstrated that of the in cells resulted in an in phosphorylation of both tyrosine residues in the C-terminal regulatory site and in the activation of Lyn was suppressed in the cells, although both of the tyrosine residues were S. H. M. H. Yamamura H. T. J. Biol. Chem. Scholar). In mast cells, studies demonstrated that is not for FcϵRI-mediated degranulation J. Siraganian R.P. J. Immunol. 1999; Scholar). These that is a for Lyn to by dephosphorylation of both autophosphorylation and C-terminal regulatory to an intramolecular interaction (11Turner H. Kinet J.P. Nature. 1999; 402: B24-B30Google Scholar, S. H. M. H. Yamamura H. T. J. Biol. Chem. Scholar). we have demonstrated that 3BP2 is a ligand of SH3/SH2 domains of of the function of Lyn by its SH3 domain binding the of the Lyn-SH3 domain in mast cell activation T. 1997; Scholar). analysis by that 3BP2 is in the plasma membrane with Lyn to stimulation In the the Lyn-SH3 domain be associated with the proline-rich region of 3BP2. is that 3BP2 with the molecule its proline-rich region, which for the and activation of Lyn FcϵRI aggregation. phosphorylation of Tyr446 create a high affinity binding site to the Lyn-SH2 and this binding the change of Lyn, to the autophosphorylation. Lyn is a PTK to Tyr446 of 3BP2. Our results suggest the that Lyn could be of some other the of mast cell Lyn its kinase for the the activation of Lyn was by the phosphorylation of C-terminal tyrosine by Lyn with a tyrosine phosphorylation is S. H. M. H. Yamamura H. T. J. Biol. Chem. Scholar). some of the activated Lyn was by by after FcϵRI K. and H. we that 3BP2 be of the regulatory of the Lyn activation cycle in FcϵRI signaling. the of 3BP2 has not been there is evidence that of the 3BP2 the is an by multiple in the and the and The are with a cells. of of this a in of the 3BP2 in of the 3BP2 protein J. H. M. J.B. S. E. 2001; Scholar). 3BP2 could be in the of and that are essential for the in have not yet been it is that function of 3BP2 could be by identified are located 3BP2 was in and the role of 3BP2 in cell signaling (2Deckert M. Tartare-Deckert S. Hernandez J. Rottapel R. Altman A. Immunity. 1998; 9: 595-605Google Scholar, I. Liu Y.C. Bernard A. Deckert M. J. Biol. Chem. 2003; 278: 7146-7153Google Scholar). to the of 3BP2 observed in have not yet been The present study has demonstrated that 3BP2 with Lyn and may the of In to signaling molecule the an protein 3BP2 has a regulatory role in PTK as a ligand binding to the SH3/SH2 domains of Lyn, similar to the function of FcϵRIγ as a ligand of Syk PTK in mast cells. We P. Siraganian and Altman for the We also for and for the