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• W2032056136 abstract "Syk (p72syk) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as an essential component of the signal transduction machinery coupled to the B-cell antigen receptor. Syk is recruited to the receptor when it is cross-linked and, in response, becomes tyrosine-phosphorylated and activated before it dissociates from the receptor and appears in the cytoplasm. To begin to explore how tyrosine phosphorylation affects Syk activation and receptor binding, Tyr-130, which is localized within the Syk inter-Src homology 2 domain region, was substituted with Phe or Glu. Substitution of Tyr-130 with Phe enhanced the binding of Syk to the receptor and increased receptor-mediated protein tyrosine phosphorylation, while substitution with Glu greatly reduced this interaction. Replacement of Tyr-130 with Glu also increased the basal activity of the kinase, while replacement with Phe decreased its activity and uncoupled kinase activation from receptor engagement. These data suggest that the phosphorylation of Tyr-130 normally plays an important role in mediating both the activation of Syk and its release from the antigen receptor. Syk (p72syk) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as an essential component of the signal transduction machinery coupled to the B-cell antigen receptor. Syk is recruited to the receptor when it is cross-linked and, in response, becomes tyrosine-phosphorylated and activated before it dissociates from the receptor and appears in the cytoplasm. To begin to explore how tyrosine phosphorylation affects Syk activation and receptor binding, Tyr-130, which is localized within the Syk inter-Src homology 2 domain region, was substituted with Phe or Glu. Substitution of Tyr-130 with Phe enhanced the binding of Syk to the receptor and increased receptor-mediated protein tyrosine phosphorylation, while substitution with Glu greatly reduced this interaction. Replacement of Tyr-130 with Glu also increased the basal activity of the kinase, while replacement with Phe decreased its activity and uncoupled kinase activation from receptor engagement. These data suggest that the phosphorylation of Tyr-130 normally plays an important role in mediating both the activation of Syk and its release from the antigen receptor. Syk (p72syk) is a 72-kDa intracellular protein-tyrosine kinase expressed in a variety of hematopoietic cells including B-cells, mast cells, macrophages, platelets, and thymocytes. Although it has been implicated in signaling through various receptor types, the role of Syk in receptor-mediated signaling has been best characterized for immune-recognition receptors such as the B-cell antigen receptor (BCR) 1The abbreviations used are: BCR, B-cell antigen receptor; SH2, Src homology 2; cfb3, cytoplasmic fragment of erythrocyte band 3; Syk(WT), Myc-tagged wild-type Syk; Syk(Y130E), Myc-tagged Syk with Tyr-130 mutated to Glu; Syk(Y130F), Myc-tagged Syk with Tyr-130 mutated to Phe; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: BCR, B-cell antigen receptor; SH2, Src homology 2; cfb3, cytoplasmic fragment of erythrocyte band 3; Syk(WT), Myc-tagged wild-type Syk; Syk(Y130E), Myc-tagged Syk with Tyr-130 mutated to Glu; Syk(Y130F), Myc-tagged Syk with Tyr-130 mutated to Phe; PAGE, polyacrylamide gel electrophoresis. (1Chan A.C. Shaw A.S. Curr. Opin. Immunol. 1996; 8: 394-401Crossref PubMed Scopus (170) Google Scholar, 2Bolen J.B. Curr. Opin. Immunol. 1995; 7: 306-311Crossref PubMed Scopus (104) Google Scholar, 3Cambier J.C. Pleiman C.M. Clark M.R. Annu. Rev. Immunol. 1994; 12: 457-486Crossref PubMed Scopus (392) Google Scholar, 4Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1952) Google Scholar). Aggregation of these receptors results in an increase in the tyrosine phosphorylation of cellular proteins that are commonly involved in signaling, increased inositol 1,4,5-trisphosphate production, calcium mobilization, and activation of the mitogen-activated protein kinase pathway (5DeFranco A.L. Curr. Opin. Immunol. 1994; 6: 364-371Crossref PubMed Scopus (30) Google Scholar). In syk −/− mice, B-cell and pre-B-cell antigen receptors fail to deliver signals for cellular survival and expansion leading to a failure of B-cell maturation, indicating a critical role for Syk in B-cell signaling (6Turner M. Mee P.J. Costello P.S. Williams O. Price A.A. Duddy L.P. Furlong M.T. Geahlen R.L. Tybulewicz V.L. Nature. 1995; 378: 298-302Crossref PubMed Scopus (645) Google Scholar, 7Cheng A.M. Rowley R.B. Pao W. Hayday A. Bolen J.B. Pawson T. Nature. 1995; 378: 303-306Crossref PubMed Scopus (548) Google Scholar). Signaling through the BCR requires the phosphorylation of a pair of uniformly spaced tyrosines in the cytoplasmic domains of the Ig-α (CD79α) and Ig-β (CD79β) subunits. These tyrosines are present within an immunoreceptor tyrosine-based activation motif, which consists of two YXX(L/I) cassettes separated by 6–8 amino acids (4Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1952) Google Scholar, 8Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1164) Google Scholar). These phosphorylated immunoreceptor tyrosine-based activation motif tyrosines serve as a docking site for the two tandem Src homology 2 (SH2) domains of Syk (9Burg D.L. Furlong M.T. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1994; 269: 28136-28142Abstract Full Text PDF PubMed Google Scholar, 10Kurosaki T. Johnson S.A. Pao L. Sada K. Yamamura H. Cambier J.C. J. Exp. Med. 1995; 182: 1815-1823Crossref PubMed Scopus (223) Google Scholar). The recruitment of Syk to the receptor results in its activation as characterized by an increase in its enzymatic activity and an increase in its phosphotyrosine content resulting from autophosphorylation and phosphorylation by a Src family protein-tyrosine kinase (11Rowley R.B. Burkhardt A.L. Chao H-G. Matsueda G.R. Bolen J.B. J. Biol. Chem. 1995; 270: 11590-11594Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 12Kurosaki T. Takata M. Yamanashi Y. Inaza T. Taniguchi T. Yamamoto T. Yamamura H. J. Exp. Med. 1994; 179: 1725-1729Crossref PubMed Scopus (250) Google Scholar). We have shown recently (13Peters J.D. Furlong M.T. Asai D.J. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1996; 271: 4755-4762Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) that the majority of activated, tyrosine-phosphorylated Syk in an anti-IgM-treated B-cell has dissociated from the receptor and is found in the soluble, cytosolic fraction. To begin to explore the role of phosphorylation in modulating the properties of Syk, we have focused on Tyr-130, an in vitro site of Syk autophosphorylation that lies within the inter-SH2 domain region (14Furlong M.T. Mahrenholz A.M. Kim K-H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). In this study, we provide evidence that phosphorylation of Syk at Tyr-130 modulates not only its ability to interact with the antigen receptor but also its intrinsic activity. Syk− DT40 chicken B-cells (15Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (587) Google Scholar) were obtained from Dr. Tomohiro Kurosaki. Unconjugated and fluorescein isothiocyanate-conjugated goat anti-chicken IgM were purchased from Bethyl Laboratories, Inc. Anti-phosphotyrosine and anti-Syk antisera have been described previously (9Burg D.L. Furlong M.T. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1994; 269: 28136-28142Abstract Full Text PDF PubMed Google Scholar, 16Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1992; 267: 8613-8619Abstract Full Text PDF PubMed Google Scholar). The 9E10 anti-Myc hybridoma cell line was purchased from ATCC, and ascites fluid was prepared by the Purdue University Cancer Center Antibody Production Facility. The cytoplasmic fragment of human erythrocyte band 3 (cfb3) was obtained from Dr. Philip Low, Purdue University. The cloning of murinesyk cDNA has been described elsewhere (14Furlong M.T. Mahrenholz A.M. Kim K-H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). To generate Myc epitope-tagged Syk, oligonucleotides containing the sense and antisense sequence of the Myc epitope along with a stop codon were annealed and then ligated to the syk cDNA. This cDNA was subcloned into the XhoI site of the pGEM/EPB expression vector, which contains a heavy chain enhancer/promoter cassette for B-cell-specific expression (17Dildrop R. Ma A. Zimmerman K. Hsu E. Tesfaye A. DePinho R. Alt F.W. EMBO J. 1989; 8: 1121-1128Crossref PubMed Scopus (81) Google Scholar). Site-directed mutagenesis was carried out using the Transformer mutagenesis kit (CLONTECH) and confirmed by DNA sequencing. Syk− DT40 cells were electroporated with 25 μg of the various Syk-Myc DNA-containing plasmids and 2.5 μg of p3'SS (Stratagene), which contains a hygromycin resistance gene. Cells were selected in hygromycin (2 mg/ml) and were screened for Syk expression by both immune complex kinase assays and Western blotting. Clones expressing comparable levels of Myc-tagged wild-type Syk (Syk(WT)), Myc-tagged Syk with Tyr-130 mutated to Glu (Syk(Y130E)), and Myc-tagged Syk with Tyr-130 mutated to Phe (Syk(Y130F)) were selected. DT40 cells were activated on ice with 25 μg/ml goat anti-chicken IgM for 15 min at 4 °C. Activation was terminated by the addition of lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Brij 96, 5 mm EDTA, 1 mm sodium orthovanadate, and 10 μg/ml each leupeptin and aprotinin). After incubation on ice for 15 min, nuclei and unbroken cells were removed by centrifugation at 15,000 × g for 5 min at 4 °C. Procedures for immunoprecipitations and immune complex kinase assays have been previously described in detail (9Burg D.L. Furlong M.T. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1994; 269: 28136-28142Abstract Full Text PDF PubMed Google Scholar). Briefly, the Myc epitope-tagged Syk proteins were immunoprecipitated with anti-Myc epitope monoclonal antibodies coupled to protein A-Sepharose (Sigma). Syk activity was detected in the immune complexes by Western blotting with anti-Syk, anti-phosphotyrosine antibodies, or by autophosphorylation in the presence of [γ-32P]ATP (5 μm). Where indicated, cfb3 (3 μg) was included as an exogenous substrate. Phosphoproteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and treated with 1 nKOH at 55 °C for 2 h. Phosphotyrosine-containing proteins were detected by autoradiography. Anti-Myc epitope immune complexes obtained from lysates of Syk(WT)-expressing DT40 cells were phosphorylatedin vitro with [γ-32P]ATP (5 μm) for the times indicated. The phosphorylated kinase was isolated by SDS-PAGE, transferred to nitrocellulose, detected by autoradiography, excised from the membrane, and incubated withl-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin as described previously (14Furlong M.T. Mahrenholz A.M. Kim K-H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). Tryptic phosphopeptides were compared by 40% polyacrylamide alkaline gel electrophoresis to a set of standard phosphopeptides derived from in vitroautophosphorylated glutathione S-transferase-Syk (14Furlong M.T. Mahrenholz A.M. Kim K-H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). These standard phosphopeptides were isolated by high performance liquid chromatography and identified by a combination of Edman sequencing and mass spectrometry as described previously (14Furlong M.T. Mahrenholz A.M. Kim K-H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). Relative levels of each phosphopeptide were determined by densitometric analysis of autoradiograms of the dried alkaline gels. Prolonged incubation (30 min) of Syk in vitro with ATP leads to its autophosphorylation on multiple sites including Tyr-130, which lies between the amino-terminal tandem pair of SH2 domains (14Furlong M.T. Mahrenholz A.M. Kim K-H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). To determine if Tyr-130 represented a major early site of autophosphorylation, the sites on Syk that become phosphorylated at shorter times of incubation with ATP were examined. For these studies, Syk bearing a Myc epitope tag at the extreme carboxyl terminus, Syk(WT), was expressed in Syk− DT40 B-cells. Syk(WT) was immunoprecipitated and autophosphorylated in vitro with [γ-32P]ATP for varying periods of time. Tryptic phosphopeptides generated by complete digestion of phospho-Syk(WT) were separated by electrophoresis on 40% polyacrylamide alkaline gels and identified by comparison with a series of known phosphopeptides (14Furlong M.T. Mahrenholz A.M. Kim K-H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). The relative extents of phosphorylation of the various sites as a function of time are illustrated in Fig. 1. The most rapidly phosphorylated residues were Tyr-317 and -130. To explore the role of Tyr-130 in Syk-receptor interactions, additional DT40 cell lines were prepared that lacked endogenous Syk but expressed mutant forms of epitope-tagged Syk in which Tyr-130 was replaced by either Phe, to prevent phosphorylation at this site, or Glu, to position a negatively charged amino acid at this site. Phosphopeptide mapping studies ofin vitro autophosphorylated Syk(Y130F) and Syk(Y130E) mutants confirmed the absence of phosphate at Tyr-130. Lack of phosphate at this site did not, however, preclude autophosphorylation at the other sites (data not shown). To compare the basal activity of Syk(Y130F) and Syk(Y130E) to that of Syk(WT), the three kinases were immunoprecipitated individually from transfected DT40 cells and assayed in the resulting immune complexes for phosphorylation of the Syk substrate, cfb3. As shown in Fig.2 A, the intrinsic activity of Syk(Y130F) was lower than that of Syk(WT) (1.3-fold). In contrast, the basal activity of Syk(Y130E) was substantially higher (2-fold) than that of Syk(WT). Furthermore, receptor cross-linking resulted in an increase (2.2-fold) in the intrinsic kinase activity of the recovered Syk(WT), but either had no effect or decreased the activity of the mutant kinases (Fig.2 B). Western blotting analyses with anti-Syk antibodies confirmed that equivalent levels of each kinase were present in the anti-Myc epitope immune complexes prepared from the untreated or anti-IgM-treated Syk-expressing DT40 cell lines (Fig.2 C). To explore the effect of Tyr-130 mutations on Syk-receptor interactions, intact BCR complexes were immunoprecipitated from Brij 96 lysates of the DT40-derived cell lines expressing wild type or mutant forms of Syk. The presence of BCR-associated kinases was detected by immune complex kinase assays. As shown in Fig. 3 A, a low level of receptor-associated Syk(WT) autophosphorylating activity could be observed co-immunoprecipitating with the clustered antigen receptor. The level of receptor-associated Syk(Y130F) activity was significantly higher than that of Syk(WT). In contrast, little or no receptor-associated Syk(Y130E) could be detected. Similar amounts of the 53- and 56-kDa forms of Lyn (as determined by Lyn autophosphorylating activity) were recruited to the clustered antigen receptors regardless of the nature of the Syk kinase expressed in the cell. This level of Lyn was similar to that observed associating with the cross-linked receptor from Syk-negative DT40 cells (data not shown). The third, and smallest, of the major proteins phosphorylated in these complexes has not been identified but, by analogy to anti-IgM immune complexes from human or murine cells, likely represents chicken Ig-α. To determine if the different levels of receptor-associated Syk activity were due to differences in the level of Syk protein bound to the receptor, anti-IgM immune complexes were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with anti-Syk antibodies. As shown in Fig. 3 B, Syk(WT) protein was found in the anti-IgM immune complexes prepared from anti-IgM-treated cells. The level of Syk(Y130F) associated with the receptor following receptor cross-linking was consistently higher than that of Syk(WT). No Syk(Y130E) protein could be detected in anti-IgM immunoprecipitates isolated from Syk(Y130E)-expressing cells. DT40 cells expressing Syk(WT), Syk(Y130F), or Syk(Y130E) were compared for their abilities to support receptor-mediated protein tyrosine phosphorylation. Flow cytometric analyses of BCR expression using fluorescein isothiocyanate-conjugated anti-IgM antibodies did not reveal any significant differences in the surface receptor expression between the three cell lines (data not shown). Lysates from resting or anti-IgM-treated cells were analyzed by immunoblotting with anti-phosphotyrosine antibodies (Fig.4 A). Receptor engagement in Syk(WT)-expressing cells resulted in the increased tyrosine phosphorylation of multiple intracellular proteins. Phosphorylation of these proteins was enhanced in cells expressing the Syk(Y130F) mutant. In contrast, there was a marked decrease in phosphotyrosine-containing proteins in activated cells expressing the Syk(Y130E) mutant. A closer examination of the differences between two additional clones of cells expressing Syk(WT) and Syk(Y130F) indicated that both the rate and extent of receptor-mediated tyrosine phosphorylation was increased in the Syk(Y130F)-expressing cells (Fig. 4 C). To characterize the state of Syk tyrosine phosphorylation in response to BCR activation, the three DT40 clones were treated with increasing concentrations of the activating anti-IgM antibody. Anti-Myc epitope immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine antibody. In the absence of receptor cross-linking, only Syk(Y130E) contained phosphotyrosine at detectable levels (Fig. 4 B). Following receptor engagement, both Syk(WT) and Syk(Y130F) were extensively phosphorylated while Syk(Y130E) exhibited only a modest increase in phosphotyrosine content (Fig.4 B). The activation of Syk in B-cells takes place at the site of clustered antigen receptors and is accompanied by a substantial increase in the phosphotyrosine content of the kinase (11Rowley R.B. Burkhardt A.L. Chao H-G. Matsueda G.R. Bolen J.B. J. Biol. Chem. 1995; 270: 11590-11594Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 16Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1992; 267: 8613-8619Abstract Full Text PDF PubMed Google Scholar, 18Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1991; 266: 14846-14849Abstract Full Text PDF PubMed Google Scholar). The activated tyrosine-phosphorylated kinase, however, dissociates from the antigen receptor, since it is found primarily in the soluble fraction of cells that have been lysed by Dounce homogenization (13Peters J.D. Furlong M.T. Asai D.J. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1996; 271: 4755-4762Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Data presented in this study indicate that the phosphorylation of Syk on Tyr-130 is likely to enhance this dissociation and is also involved in the receptor-mediated activation of the kinase. Syk has multiple tyrosine residues that are potential sites of phosphorylation. Tyrosine residues 130, 290, 317, 342, 346, 358, 519, 520, 623, and 624 are all phosphorylated when Syk is incubated in vitro with ATP (14Furlong M.T. Mahrenholz A.M. Kim K-H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). Based on previous studies of Syk and by analogy to the Syk homolog, ZAP-70, some phosphorylation sites are likely to regulate the catalytic activity of Syk (Tyr-519 and -520) (10Kurosaki T. Johnson S.A. Pao L. Sada K. Yamamura H. Cambier J.C. J. Exp. Med. 1995; 182: 1815-1823Crossref PubMed Scopus (223) Google Scholar, 19Kong G. Dalton M. Wardenburg J.B. Straus D. Kurosaki T. Chan A.C. Mol. Cell. Biol. 1996; 16: 5026-5035Crossref PubMed Scopus (120) Google Scholar, 20Wange R.L. Guitian R. Isakov N. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 18730-18733Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) while others form docking sites for SH2 domain-containing proteins such as phospholipase C-γ1 (Tyr-342 and/or -346) (21Law C.-L. Chandran K.A. Sidorenko S.P. Clark E.A. Mol. Cell. Biol. 1996; 16: 1305-1315Crossref PubMed Google Scholar), Lck (Tyr-519 and -520) (22Couture C. Deckert M. Williams S. Russo F.O. Altman A. Mustelin T. J. Biol. Chem. 1996; 271: 24294-24299Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and others (23Neumeister E.N. Shu Y. Richard S. Terhorst C. Chan A.C. Shaw A.S. Mol. Cell. Biol. 1995; 15: 3171-3178Crossref PubMed Google Scholar). None of the previously characterized sites, however, have been implicated in mediating kinase-receptor interactions. We focused our attention on Tyr-130 since it is a prominent and early site of Syk autophosphorylation (Fig. 1) and is localized within the inter-SH2 domain region. Several observations are consistent with a role for Tyr-130 phosphorylation in modulating the interactions of Syk with the antigen receptor. First, if the phosphorylation of Tyr-130 is prevented by its replacement with Phe, receptor binding is enhanced. Low levels of Syk(WT) activity or protein can be detected binding to the aggregated antigen receptor (Fig. 3), consistent with previous observations (9Burg D.L. Furlong M.T. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1994; 269: 28136-28142Abstract Full Text PDF PubMed Google Scholar,10Kurosaki T. Johnson S.A. Pao L. Sada K. Yamamura H. Cambier J.C. J. Exp. Med. 1995; 182: 1815-1823Crossref PubMed Scopus (223) Google Scholar). It is likely that this interaction of Syk with the antigen receptor is transient in nature. Functional coupling of Syk(WT) to the antigen receptor is also indicated since anti-IgM antibodies induce Syk(WT) phosphorylation (Fig. 4 B), Syk(WT) activation (Fig.2 B), and the phosphorylation of intracellular proteins (Fig.4, A and C). When Tyr-130 is replaced by Phe to prevent phosphorylation at this site, the level of receptor-associated kinase that can be recovered increases significantly (Fig. 3). This increased association of Syk(Y130F) with the receptor also enhances the receptor-mediated phosphorylation of intracellular targets (Fig. 4,A and C). On the other hand, when Tyr-130 is replaced with Glu, no receptor-associated kinase can be detected in immunoprecipitated BCR complexes from anti-IgM-activated cells (Fig.3). As a result, the receptor-mediated phosphorylation of intracellular proteins on tyrosine is greatly diminished in the Syk(Y130E)-expressing cells (Fig. 4 A). The receptor-mediated increases in tyrosine phosphorylation that do remain in Syk(Y130E)-expressing cells likely result either from a transient or low affinity interaction of Syk(Y130E) with the receptor or from an interaction of Syk(Y130E) with Lyn, which is still activated by receptor engagement in these cells. Several observations also indicate that the phosphorylation of Tyr-130 may be an important step in the activation of Syk. When Tyr-130 is replaced by Phe, the intrinsic activity of the kinase is reduced compared with that of the wild-type enzyme (Fig. 2 A). More significantly, we have been unable to observe a receptor-coupled activation of Syk(Y130F) (Fig. 2 B) despite the enhanced ability of the enzyme to bind aggregated receptors (Fig.3 A). Either the Syk(Y130F) kinase is not activated by receptor engagement or the percentage of the expressed enzyme that is activated is below our detection limits. If Tyr-130 is replaced by Glu to permanently position a negatively charged residue at this site, the kinase now exhibits a basal activity twice that of Syk(WT) and nearly three times that of Syk(Y130F) (Fig. 2 A). It is interesting to note that the phosphorylation of downstream target proteins on tyrosine in response to receptor aggregation appears more dependent on the length of time Syk remains associated with the receptor than on increases in its intrinsic activity (Fig. 4, A andC). The mechanisms by which the phosphorylation of Tyr-130 alters Syk activity and receptor binding have yet to be determined. By analogy to Tyr-126 of the Syk-family kinase, ZAP-70, Tyr-130 would be expected to lie near the apex of a coiled coil of α-helices located within the inter-SH2 domain region (24Hatada M.H. Lu X. Laird E.R. Green J. Morgenstern J.P. Lou M. Marr C.S. Phillips T.B. Ram M.K. Theriault K. Zoller M.J. Karas J.L. Nature. 1995; 377: 32-38Crossref PubMed Scopus (294) Google Scholar). It has been hypothesized that this region functions to properly position the SH2 domains in an orientation appropriate for the inter-SH2 domain interactions required for binding a dually phosphorylated immunoreceptor tyrosine-based activation motif (24Hatada M.H. Lu X. Laird E.R. Green J. Morgenstern J.P. Lou M. Marr C.S. Phillips T.B. Ram M.K. Theriault K. Zoller M.J. Karas J.L. Nature. 1995; 377: 32-38Crossref PubMed Scopus (294) Google Scholar). This region may also be in a position to mediate intramolecular interactions between the coiled coil domain and the kinase active site (24Hatada M.H. Lu X. Laird E.R. Green J. Morgenstern J.P. Lou M. Marr C.S. Phillips T.B. Ram M.K. Theriault K. Zoller M.J. Karas J.L. Nature. 1995; 377: 32-38Crossref PubMed Scopus (294) Google Scholar). The amino acids surrounding Tyr-130 share the sequence of LXX(E/D)Y with Syk phosphorylation sites on α-tubulin and erythrocyte band 3, proteins that physically associate with Syk (13Peters J.D. Furlong M.T. Asai D.J. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1996; 271: 4755-4762Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar,25Harrison M.L. Isaacson C.C. Burg D.L. Geahlen R.L. Low P.S. J. Biol. Chem. 1994; 269: 955-959Abstract Full Text PDF PubMed Google Scholar). On band 3, this site forms a loop that surrounds the tyrosine and serves as a site involved in protein/protein associations (26Schneider M.L. Post C.B. Biochemistry. 1995; 34: 16574-16584Crossref PubMed Scopus (25) Google Scholar). Phosphorylation of this tyrosine destabilizes the loop (26Schneider M.L. Post C.B. Biochemistry. 1995; 34: 16574-16584Crossref PubMed Scopus (25) Google Scholar) and disrupts these interactions (27Low P.S. Allen D.P. Zioncheck T.F. Chari P. Willardson B.M. Geahlen R.L. Harrison M.L. J. Biol. Chem. 1987; 262: 4592-4596Abstract Full Text PDF PubMed Google Scholar). By analogy, phosphorylation of Tyr-130 might reasonably be expected to cause changes in the local conformation of the inter-SH2 domain region that alters both the SH2-SH2 interface interactions and the interactions between the inter-SH2 region and the kinase active site. Thus phosphorylation of Tyr-130 could negatively influence the binding of Syk to the antigen receptor and at the same time allow increased access of the catalytic site to protein substrates. Further experiments on the structure and function of Syk will be required to formally validate this model. We thank Dr. Tomohiro Kurosaki for the generous gift of the Syk− DT40 cell line and Dr. Frederick W. Alt for the pGEM/EPB expression vector." @default.
• W2032056136 created "2016-06-24" @default.
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• W2032056136 date "1997-04-01" @default.
• W2032056136 modified "2023-10-10" @default.
• W2032056136 title "Syk Activation and Dissociation from the B-cell Antigen Receptor Is Mediated by Phosphorylation of Tyrosine 130" @default.
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• W2032056136 doi "https://doi.org/10.1074/jbc.272.16.10377" @default.
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