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• W2005857763 abstract "Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FcεRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FcεRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FcεRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivoand in vitro. In FcεRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation. Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FcεRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FcεRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FcεRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivoand in vitro. In FcεRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation. immunoreceptor tyrosine-based activation motif monoclonal antibody specific anti-Syk activation loop phosphotyrosine antibody Aggregation of the high affinity IgE receptor, FcεRI, on mast cells results in release of mediators that initiate inflammatory and allergic responses (1Beaven M.A. Metzger H. Immunol. Today. 1993; 14: 222-226Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 2Siraganian R.P. Middleton E.J. Reed C.E. Ellis E.F. Adkinson N.F.J. Yunginger J.W. Busse W.W. Allergy: Principles and Practice. Mosby-Year Book, Inc., St. Louis, MO1993: 105-134Google Scholar). The signal transduction pathway initiated by this receptor has many similarities to those of antigen receptors on T or B cells. These immune receptors lack intrinsic tyrosine kinase activity but possess signal-transducing subunits that contain the immunoreceptor tyrosine-based activation motif (ITAM)1 (3Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1167) Google Scholar, 4Weiss A. Cell. 1993; 73: 209-212Abstract Full Text PDF PubMed Scopus (475) Google Scholar, 5Cambier J.C. J. Immunol. 1995; 155: 3281-3285PubMed Google Scholar, 6Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). Antigen stimulation induces rapid phosphorylation of tyrosines in the ITAM, which then recruit a Syk family tyrosine kinase from the cytoplasm to the membrane. The binding of Syk to the phosphorylated ITAM induces a conformational change in Syk, which results in its activation and tyrosine phosphorylation (7Hutchcroft J.E. Geahlen R.L. Deanin G.G. Oliver J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9107-9111Crossref PubMed Scopus (206) Google Scholar, 8Benhamou 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, 9Minoguchi 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, 10Kurosaki T. Takata M. Yamanashi Y. Inazu T. Taniguchi T. Yamamoto T. Yamamura H. J. Exp. Med. 1994; 179: 1725-1729Crossref PubMed Scopus (252) Google Scholar, 11Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar, 12Shiue 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, 13Rowley 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, 14Shiue L. Zoller M.J. Brugge J.S. J. Biol. Chem. 1995; 270: 10498-10502Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 15Kimura T. Sakamoto H. Appella E. Siraganian R.P. Mol. Cell. Biol. 1996; 16: 1471-1478Crossref PubMed Scopus (101) Google Scholar, 16Chen T. Repetto B. Chizzonite R. Pullar C. Burghardt C. Dharm E. Zhao A.C. Carroll R. Nunes P. Basu M. Danho W. Visnick M. Kochan J. Waugh D. Gilfillan A.M. J. Biol. Chem. 1996; 271: 25308-25315Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Accumulated data suggests that Syk is essential in mediating FcεRI signaling, which is further confirmed by the experiments using Syk-deficient mast cells (17Rivera V.M. Brugge J.S. Mol. Cell. Biol. 1995; 15: 1582-1590Crossref PubMed Google Scholar, 18Taylor J.A. Karas J.L. Ram M.K. Green O.M. Seidel-Dugan C. Mol. Cell. Biol. 1995; 15: 4149-4157Crossref PubMed Google Scholar, 19Scharenberg A.M. Lin S. Cuenod B. Yamamura H. Kinet J.P. EMBO J. 1995; 14: 3385-3394Crossref PubMed Scopus (143) Google Scholar, 20Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 21Zhang J. Berenstein E.H. Evans R.L. Siraganian R.P. J. Exp. Med. 1996; 184: 71-79Crossref PubMed Scopus (239) Google Scholar, 22Costello P.S. Turner M. Walters A.E. Cunningham C.N. Bauer P.H. Downward J. Tybulewicz V.L.J. Oncogene. 1996; 13: 2595-2605PubMed Google Scholar). In these Syk-deficient cells, antigen stimulation does not induce cellular protein tyrosine phosphorylation, calcium mobilization, or degranulation, and these defects are reconstituted by the expression of Syk. Syk family tyrosine kinases are also important for signaling from other immunoreceptors such as the Fcγ receptors and T or B cell receptors (23Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (588) Google Scholar, 24Cheng A.M. Rowley B. Pao W. Hayday A. Bolen J.B. Pawson T. Nature. 1995; 378: 303-306Crossref PubMed Scopus (549) Google Scholar, 25Turner 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 (648) Google Scholar, 26Crowley M.T. Costello P.S. Fitzer-Attas C.J. Turner M. Meng F.Y. Lowell C. Tybulewicz V.J. DeFranco A.L. J. Exp. Med. 1997; 186: 1027-1039Crossref PubMed Scopus (409) Google Scholar, 27Turner M. Gulbranson-Judge A. Quinn M.E. Walters A.E. MacLennan I.M. Tybulewicz V.J. J. Exp. Med. 1997; 186: 2013-2021Crossref PubMed Scopus (144) Google Scholar, 28Kiefer F. Brumell J. Al-Alawi N. Latour S. Cheng A. Veillette A. Grinstein S. Pawson T. Mol. Cell. Biol. 1998; 18: 4209-4220Crossref PubMed Google Scholar). Phosphopeptide mapping has identified 10 tyrosine residues in Syk that are autophosphorylated after an in vitro kinase reaction (29Furlong M.T. Mahrenholz A.M. Kim K.H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta Mol. Cell Res. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). Phosphorylation at these tyrosine residues may provide binding sites for substrates or may be important for regulating the catalytic activity of Syk. Indeed, phosphorylation of Tyr317 of Syk negatively down-regulates signal transduction in mast cells, probably by a pathway that involves Cbl, a negative regulator of protein tyrosine kinases (30Lupher Jr., M.L. Rao N. Lill N.L. Andoniou C.E. Miyake S. Clark E.A. Druker B. Band H. J. Biol. Chem. 1998; 273: 35273-35281Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 31Deckert M. Elly C. Altman A. Liu Y.C. J. Biol. Chem. 1998; 273: 8867-8874Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 32Keshvara L.M. Isaacson C.C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar, 33Sada K. Zhang J. Siraganian R.P. J. Immunol. 2000; 164: 338-344Crossref PubMed Scopus (56) Google Scholar). Similarly, the tyrosine residues in the activation loop of Syk (Tyr519 and Tyr520 in rat Syk) are essential for propagating downstream signaling events (34Kurosaki T. Johnson S.A. Pao L. Sada K. Yamamura H. Cambier J.C. J. Exp. Med. 1995; 182: 1815-1823Crossref PubMed Scopus (224) Google Scholar, 35El-Hillal O. Kurosaki T. Yamamura H. Kinet J.P. Scharenberg A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1919-1924Crossref PubMed Scopus (108) Google Scholar, 36Zhang J. Kimura T. Siraganian R.P. J. Immunol. 1998; 161: 4366-4374PubMed Google Scholar). These results suggest that tyrosine phosphorylation is a critical feature for regulating Syk enzymatic activity and for its function. Structural studies suggest that activation loop tyrosines are important for regulating the enzymatic activity of many kinases. In the nonactivated state, these tyrosines occupy the catalytic center and hinder ATP and/or substrate binding. Phosphorylation of one or both of the tyrosines induces a conformational change that moves the loop out of the catalytic site, thereby allowing the binding of ATP and/or substrate. Therefore, the phosphorylation of these activation loop tyrosines is thought to regulate enzymatic activation. The crystal structure of the enzymatic domain of Syk or ZAP70 has not been established, although data suggest that a similar model also applies to these kinases. For example, mutants of Syk with the activation loop tyrosines mutated fail to signal from antigen receptors although there is no loss of in vitro enzymatic activity (34Kurosaki T. Johnson S.A. Pao L. Sada K. Yamamura H. Cambier J.C. J. Exp. Med. 1995; 182: 1815-1823Crossref PubMed Scopus (224) Google Scholar, 36Zhang J. Kimura T. Siraganian R.P. J. Immunol. 1998; 161: 4366-4374PubMed Google Scholar). There are still unresolved questions in understanding the relationship of Syk tyrosine phosphorylation with its function. For example, in the RBL-2H3 mast cell line both FcεRI stimulation and the cell surface binding of anti-ganglioside mAb AA4 induce similar levels of Syk tyrosine phosphorylation. However, the response of the cell to these two stimulants is dramatically different. Whereas FcεRI aggregation results in degranulation, mAb AA4 binding induces only minor, if any, histamine release (37Swaim W.D. Minoguchi K. Oliver C. Hamawy M.M. Kihara H. Stephan V. Berenstein E.H. Siraganian R.P. J. Biol. Chem. 1994; 269: 19466-19473Abstract Full Text PDF PubMed Google Scholar). Therefore, examination of the phosphorylation of individual tyrosine residues will be necessary to better understand the function of Syk and the signal transduction pathways. In the present study, a polyclonal antibody was developed that specifically detected the phosphorylation of the Syk activation loop tyrosines. This antibody, in combination with synthetic phosphopeptides, can distinguish the phosphorylation status of each of the two adjacent tyrosine residues. We compared the FcεRI- and mAb AA4-induced histamine release with the total and activation loop tyrosine phosphorylation of Syk. The results demonstrate that Syk activation loop tyrosine phosphorylation is directly related to the capacity of Syk to propagate downstream intracellular signals. Our experiments also suggest that, in vivo, both tyrosine residues in the Syk activation loop were phosphorylated after FcεRI stimulation. Furthermore, Syk kinase activity played the major role in phosphorylating these two tyrosines. Triton X-100 and protein A-agarose beads were obtained from Sigma. The materials for electrophoresis were from Novex (San Diego, CA). The horseradish peroxidase-conjugated anti-phosphotyrosine antibody 4G-10 was from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-ganglioside monoclonal antibody AA4, mouse and rabbit anti-Syk antibody, and other materials not indicated were as described previously (21Zhang J. Berenstein E.H. Evans R.L. Siraganian R.P. J. Exp. Med. 1996; 184: 71-79Crossref PubMed Scopus (239) Google Scholar, 38Basciano L.K. Berenstein E.H. Kmak L. Siraganian R.P. J. Biol. Chem. 1986; 261: 11823-11831Abstract Full Text PDF PubMed Google Scholar). The rabbit anti-Syk antibody was raised to a sequence in the linker region between the second SH2 domain and the kinase region and has been characterized previously (8Benhamou 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). Peptides were synthesized using a Milligen model 9500 peptide synthesizer. The parent peptide NH2-ALRADENYYKAQTHGC-COOH, corresponding to the activation loop of rat Syk amino acid sequence 512–526, was made using 9-fluorenylmethoxycarbony chemistry, with a cysteine COOH-terminal resin. For mono- and diphosphorylated peptides, a phosphorylated derivative of tyrosine was used instead of Tyr. Rabbits were immunized with the conjugate of the diphosphorylated peptide coupled to keyhole limpet hemocyanin (Sigma) via the COOH-terminal cysteine residue usingm-maleimido-benzoyl-N-hydroxysuccinimide as a cross-linking reagent. The phosphopeptide-specific sera were purified by negative absorption with CH-Sepharose affinity beads containing the native, nonphosphorylated peptide. Rat basophilic leukemia RBL-2H3 cells, Syk-negative variant TB1A2 cells, and their wild-type or activation loop tyrosine mutant transfectants (Y519F mutant, Y520F mutant, and Y519F/Y520F mutant) have been described previously (21Zhang J. Berenstein E.H. Evans R.L. Siraganian R.P. J. Exp. Med. 1996; 184: 71-79Crossref PubMed Scopus (239) Google Scholar, 36Zhang J. Kimura T. Siraganian R.P. J. Immunol. 1998; 161: 4366-4374PubMed Google Scholar). A point mutation of Lys396 to Arg (dead kinase) was prepared by using a polymerase chain reaction-based method and confirmed by DNA sequencing. The dead kinase cDNA was cloned into pSVL expression vector and stably expressed in Syk-negative TB1A2 cells as described previously (21Zhang J. Berenstein E.H. Evans R.L. Siraganian R.P. J. Exp. Med. 1996; 184: 71-79Crossref PubMed Scopus (239) Google Scholar). For FcεRI activation, the monolayers were cultured overnight either without or with antigen-specific IgE. The cells cultured with IgE were stimulated with the antigen at concentrations from 0.01 to 1.0 μg/ml. In some experiments, cells cultured without IgE were stimulated with different concentrations of AA4 antibody. After stimulation for the indicated times, the supernatants were removed for histamine analysis. For pervanadate stimulation, cells cultured without IgE were stimulated with 0.4 mm pervanadate for 30 min at 37 °C. After stimulation, the cell monolayers were rinsed with ice-cold PBS containing 2 mm Na3VO4, and protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 90 milliunits/ml aprotinin, 50 μg/ml leupeptin, 50 μg/ml pepstatin) and solubilized in Triton lysis buffer (1% Triton, 20 mm Tris, pH 7.4, 100 mm NaCl, 50 mm NaF plus protease inhibitors and Na3VO4). The postnuclear supernatants were immunoprecipitated with rabbit anti-Syk antibody bound to protein A-agarose beads. After rotation at 4 °C for 1 h, the beads were washed four times with ice-cold lysis buffer, and the proteins were eluted by boiling for 5 min with SDS-polyacrylamide gel electrophoresis sample buffer as described previously (11Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar). Whole cell lysates or immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The blots were probed with anti-phosphotyrosine mAb 4G10, anti-Syk activation loop phosphotyrosine antibody (anti-phospho-AL-Syk), or mouse anti-Syk antibody. In all blots, proteins were visualized by enhanced chemiluminescence (Renaissance). Syk immunoprecipitated from nonstimulated cells was further washed with kinase buffer (30 mm HEPES, pH 7.5, 10 mm MgCl2, and 2 mm MnCl2) and resuspended in 25 μl of this kinase buffer. The kinase reactions were at 4 °C with 5 μm ATP and with or without diphosphorylated γ-ITAM peptide in the reaction solution. The reactions were stopped by the addition of 25 μl of 2× Laemmli sample buffer and boiling for 10 min. The eluted proteins were separated under reducing conditions by SDS-polyacrylamide gel electrophoresis, electrotransferred to membranes, and blotted by anti-phosphotyrosine mAb 4G10, anti-phospho-AL-Syk, or mouse anti-Syk antibody. In all blots, proteins were visualized by enhanced chemiluminescence (Renaissance). To examine the function of tyrosine phosphorylation of the Syk activation loop, we generated an antibody against a phosphopeptide based on the activation loop sequence. The serum was adsorbed by the corresponding nonphosphorylated peptide to deplete antibodies reactive with the native activation loop tyrosines. The specificity of the antibody was tested using Syk-negative mast cells reconstituted with wild type or activation loop mutant forms of Syk (21Zhang J. Berenstein E.H. Evans R.L. Siraganian R.P. J. Exp. Med. 1996; 184: 71-79Crossref PubMed Scopus (239) Google Scholar, 36Zhang J. Kimura T. Siraganian R.P. J. Immunol. 1998; 161: 4366-4374PubMed Google Scholar). These different forms of Syk had the two tyrosines in the putative activation loop (tyrosine residues 519 and 520 of the rat sequence) mutated to phenylalanine either singly or in combination (Y519F mutant, Y520F mutant, and Y519F/Y520F mutant). The different cell lines were treated with pervanadate to induce maximum tyrosine phosphorylation of cellular proteins. Syk was then immunoprecipitated from the cell lysates with a rabbit antibody that binds a sequence in the linker region of Syk. The immunoprecipitated proteins were analyzed by immunoblotting with either mAb 4G10 that binds general phosphotyrosine residues or with the specific anti-Syk activation loop phosphotyrosine antibody (Fig.1 A). The different forms of Syk from the pervanadate treated cells gave a strong phosphorylation signal with anti-phosphotyrosine 4G10 antibody. In contrast, with the anti-phospho-AL-Syk antibody a strong signal was observed only with the wild-type Syk, while the Y519F and Y520F mutant proteins gave positive but weaker signals, and there was no signal with the Y519F/Y520F mutant. As expected, the anti-phospho-AL-Syk did not react with the nonphosphorylated Syk. Therefore, this antibody is phosphospecific for the activation loop tyrosines of Syk and does not bind to the native molecule or other phosphotyrosyl residues in Syk.Figure 1Specificity of the Syk activation loop phosphospecific antibody. A Syk-negative mast cell line stably transfected with wild type or the different Syk mutants was either not stimulated or stimulated with pervanadate for 30 min. Syk was immunoprecipitated with rabbit anti-Syk, separated by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose for immunoblot analysis. A, immuno-blotting with anti-phosphotyrosine antibody (Anti-PY), anti-phospho- AL-Syk, or anti-Syk antibody. B, immunoblotting with anti-phosphotyrosine antibody (Anti-PY), anti-phospho-AL-Syk, and anti-phospho-AL-Syk in the presence of the synthetic peptide with either the first (pYY) or the second (YpY) tyrosine phosphorylated. The peptides were used at 0.025 nm. The different forms of Syk in these cells are as follows: wild-type (WT), Y519F (FY), Y520F (YF), and Y519F/Y520F (FF) mutants.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Synthetic phosphorylated peptides with the same backbone as that used for immunization were then used to determine whether the anti-phospho-AL-Syk antibody could distinguish the phosphorylation of each of the two adjacent tyrosines in the activation loop (Fig.1 B). These phosphopeptides with either the first or the second tyrosine phosphorylated rendered the antibody specific to the reciprocal site. For example, when a peptide with the first tyrosine phosphorylated was added to the anti-phospho-AL-Syk antibody, there was a signal only when the second tyrosine of the activation loop was phosphorylated. These experiments demonstrate that anti-phospho-AL-Syk specifically detects the phosphorylation status of both activation loop tyrosines of Syk. A puzzling aspect of mast cell signal transduction is that some cell stimulants, such as the anti-cell surface ganglioside mAb AA4, induce strong tyrosine phosphorylation of cellular proteins including Syk, but not histamine release (37Swaim W.D. Minoguchi K. Oliver C. Hamawy M.M. Kihara H. Stephan V. Berenstein E.H. Siraganian R.P. J. Biol. Chem. 1994; 269: 19466-19473Abstract Full Text PDF PubMed Google Scholar). To investigate whether this is related to differences in the phosphorylation of the activation loop tyrosines, RBL-2H3 cells were stimulated by either IgE plus specific antigen or by mAb AA4 (Fig.2). As had been observed previously (37Swaim W.D. Minoguchi K. Oliver C. Hamawy M.M. Kihara H. Stephan V. Berenstein E.H. Siraganian R.P. J. Biol. Chem. 1994; 269: 19466-19473Abstract Full Text PDF PubMed Google Scholar) by using anti-general phosphotyrosine antibodies, both FcεRI-stimulation and mAb AA4 binding resulted in strong tyrosine phosphorylation of Syk. However, immunoblotting with anti-phospho-AL-Syk and analysis of histamine release detected marked differences with these two stimulants. Receptor aggregation with IgE plus antigen induced strong phosphorylation of the activation loop tyrosines and dramatic histamine release, while mAb AA4 binding only stimulated minor Syk activation loop tyrosine phosphorylation and minimal histamine release. To further confirm the relationship between Syk activation loop tyrosine phosphorylation and cell degranulation, RBL-2H3 cells were incubated with different concentrations of antigen or AA4 antibody, and histamine release, the total Syk and activation loop tyrosine phosphorylation determined (Fig.3). The results were similar; after IgE plus antigen stimulation, there was histamine release together with strong tyrosine phosphorylation signals of both whole Syk and the activation loop tyrosines. In contrast, mAb AA4 binding induced only strong tyrosine phosphorylation for whole Syk. These experiments suggest that, compared with total Syk tyrosine phosphorylation, the activation loop tyrosine phosphorylation is a better indicator for Syk functional activation that leads to degranulation. We then examined the role of Syk itself in the phosphorylation of its activation loop tyrosines. Syk was immunoprecipitated from nonstimulated RBL-2H3 cells and subjected to anin vitro kinase assay with or without diphosphorylated γ-ITAM peptide (Fig. 4). As reported previously, the immunoprecipitated Syk from these quiescent cells was not tyrosine-phosphorylated but had intrinsic kinase activity, which was enhanced by the addition of diphosphorylated γ-ITAM peptide (15Kimura T. Sakamoto H. Appella E. Siraganian R.P. Mol. Cell. Biol. 1996; 16: 1471-1478Crossref PubMed Scopus (101) Google Scholar). Immunoblotting with anti-phospho-AL-Syk indicated that Syk was able to phosphorylate its own activation loop tyrosines in the absence of other kinases. The addition of diphosphorylated γ-ITAM enhanced this function. Therefore, Syk is capable of phosphorylating its activation loop tyrosines in vitro. To further define the role of Syk for the in vivophosphorylation of its own activation loop tyrosine, cells expressing wild-type Syk or kinase-dead Syk were stimulated with IgE plus specific antigen (Fig. 5). Since the extent of the tyrosine phosphorylation of kinase-dead Syk was low, we used different amounts of the cell lysates in the immunoblots to estimate the level of phosphorylation. The receptor-induced total phosphorylation of kinase-dead Syk was approximately 20% as much as that of wild type Syk; however, the activation loop tyrosine phosphorylation of kinase-dead Syk showed a more dramatic decrease (less than 10% of wild type). Therefore, in mast cells Syk plays the major role in the receptor-induced phosphorylation of its own activation loop tyrosines, while other kinases, such as Lyn, probably make minor contributions to this phosphorylation. One of the differences between Syk and ZAP70 is that each of the two adjacent tyrosines in the activation loop of Syk is essential for propagation of receptor-induced signaling (36Zhang J. Kimura T. Siraganian R.P. J. Immunol. 1998; 161: 4366-4374PubMed Google Scholar, 39Chan A.C. Dalton M. Johnson R. Kong G.H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Crossref PubMed Scopus (325) Google Scholar, 40Wange 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). In the present study, we noticed that after pervanadate stimulation, the 519 and 520 tyrosine residues of Syk were both phosphorylated (Fig.1 B). We then investigated the FcεRI-induced phosphorylation of the two activation loop tyrosines in wild-type Syk. After IgE plus antigen stimulation, Syk was immunoprecipitated from RBL-2H3 cells and analyzed by blotting with anti-phospho-AL-Syk antibody in the presence or absence of phosphopeptides to render it site-specific (Fig. 6). Both 519pY and 520pY peptides obviously inhibited the activation loop tyrosine phosphorylation signal. The inhibition with the two peptides demonstrates that after receptor aggregation both tyrosine residues in the activation loop are phosphorylated. To study the mechanism of Syk regulation by phosphorylation, an antibody was developed that was specific for the phosphorylated tyrosines in the activation loop of Syk. By using this antibody, we found that both tyrosine residues at the Syk activation loop were phosphorylated in vivo. Syk played a major role in phosphorylating these tyrosines. In antigen-stimulated cells the FcεRI-induced phosphorylation of the activation loop tyrosines paralleled the total Syk phosphorylation, and both correlated with downstream propagation of signals for histamine release. However, the cell surface binding of anti-ganglioside mAb AA4 induced only strong total Syk tyrosine phosphorylation, minimal histamine release and weak phosphorylation of the activation loop tyrosines. Although Syk and ZAP70 belong to the same tyrosine kinase family and have many functional and structural similarities, there are differences in their signaling capacity and the mechanism of their regulation (41Couture C. Williams S. Gauthier N. Tailor P. Mustelin T. Eur. J. Biochem. 1997; 246: 447-451Crossref PubMed Scopus (30) Google Scholar, 42Williams B.L. Schreiber K.L. Zhang W.G. Wange R.L. Samelson L.E. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (224) Google Scholar, 43Fitzer-Attas C.J. Schindler D.G. Waks T. Eshhar Z. J. Immunol. 1998; 160: 145-154PubMed Google Scholar). The activation of Syk can be induced by binding to phosphorylated ITAM, while ZAP70 requires additional stimulatory input from Lck (13Rowley 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, 14Shiue L. Zoller M.J. Brugge J.S. J. Biol. Chem. 1995; 270: 10498-10502Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 44Kong G.H. Dalton M. Wardenburg J.B. Straus D. Kurosaki T. Chan A.C. Mol. Cell. Biol. 1996; 16: 5026-5035Crossref PubMed Scopus (120) Google Scholar, 45Zoller K.E. MacNeil I.A. Brugge J.S. J. Immunol. 1997; 158: 1650-1659PubMed Google Scholar, 46Isakov N. Wange R.L. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1996; 271: 15753-15761Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In the Jurkat T cell line, Syk, but not ZAP70, can transduce T cell receptor signals independent of CD45 and of Lck (47Chu D.H. Spits H. Peyron J.F. Rowley R.B. Bolen J.B. Weiss A. EMBO J. 1996; 15: 6251-6261Crossref PubMed Scopus (113) Google Scholar). Similarly, in a Syk and CD45 double deficient mast cell line, the expression of Syk but not ZAP70 reconstitutes FcεRI signaling (48Zhang J. Siraganian R.P. J. Immunol. 1999; 163: 2508-2516PubMed Google Scholar). In the in vitro experiments, we found that Syk phosphorylated its activation loop tyrosines probably by a trans-phosphorylation reaction. The addition of diphosphorylated ITAM peptides to immunoprecipitated Syk enhanced the rate of this phosphorylation. The results from the in vivo study also support this model. In FcεRI-activated cells, there was strong phosphorylation of the activation loop tyrosines of wild type Syk but only minimal phosphorylation at this site of the kinase-inactive Syk. Altogether, these results suggest that Syk plays a major role in phosphorylating its activation loop tyrosines, while other kinases, such as Lyn, are playing a minor contribution in this process (10Kurosaki T. Takata M. Yamanashi Y. Inazu T. Taniguchi T. Yamamoto T. Yamamura H. J. Exp. Med. 1994; 179: 1725-1729Crossref PubMed Scopus (252) Google Scholar). Site-directed mutagenesis of the activation loop tyrosines of ZAP70 and Syk have demonstrated another difference between these two kinases. For ZAP70, mutation of the first activation loop tyrosine to Phe increasesin vitro kinase activity and enhances the in vivoactivation signal in a Syk-negative avian B cell line, whereas ZAP70 with a mutation of the second tyrosine has normal enzymatic activity but cannot transmit antigen-receptor signaling (39Chan A.C. Dalton M. Johnson R. Kong G.H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Crossref PubMed Scopus (325) Google Scholar, 40Wange 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, 44Kong G.H. Dalton M. Wardenburg J.B. Straus D. Kurosaki T. Chan A.C. Mol. Cell. Biol. 1996; 16: 5026-5035Crossref PubMed Scopus (120) Google Scholar). In contrast, mutation of either one of these two tyrosines in Syk results in a loss of its capacity to function in the IgE-receptor signaling pathway, although the mutations have no effect on in vitroenzymatic activity (36Zhang J. Kimura T. Siraganian R.P. J. Immunol. 1998; 161: 4366-4374PubMed Google Scholar). In the present study, we found that in normal RBL-2H3 cells, both tyrosines of the activation loop were phosphorylated after IgE receptor stimulation. Since in RBL-2H3 cells IgE plus antigen induced more than 60% histamine release, it appears unlikely that the phosphorylation of any one tyrosine of the activation loop plays an inhibitory role in propagating degranulation. Therefore, this observation supported the results of functional studies of Syk activation loop mutants in which it was found that if either one of the tyrosines was mutated there was loss of the signal for degranulation. Syk contains multiple tyrosine residues, 10 of which are autophosphorylated in vitro (29Furlong M.T. Mahrenholz A.M. Kim K.H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta Mol. Cell Res. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). Mutagenesis of these different tyrosines has demonstrated that several of these residues are important in up- or down-regulating Syk function. For example, the putative Cbl interaction site, Tyr317, negatively regulates Syk signaling (30Lupher Jr., M.L. Rao N. Lill N.L. Andoniou C.E. Miyake S. Clark E.A. Druker B. Band H. J. Biol. Chem. 1998; 273: 35273-35281Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 31Deckert M. Elly C. Altman A. Liu Y.C. J. Biol. Chem. 1998; 273: 8867-8874Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 32Keshvara L.M. Isaacson C.C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar, 33Sada K. Zhang J. Siraganian R.P. J. Immunol. 2000; 164: 338-344Crossref PubMed Scopus (56) Google Scholar). Similarly, mutation of the three tyrosines at the COOH-terminal region of Syk results in a gain of function in T cell lines (49Zeitlmann L. Knorr T. Knoll M. Romeo C. Sirim P. Kolanus W. J. Biol. Chem. 1998; 273: 15445-15452Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In contrast, Tyr519 and Tyr520 in the activation loop of Syk are essential for propagating IgE-induced signals for degranulation (34Kurosaki T. Johnson S.A. Pao L. Sada K. Yamamura H. Cambier J.C. J. Exp. Med. 1995; 182: 1815-1823Crossref PubMed Scopus (224) Google Scholar, 36Zhang J. Kimura T. Siraganian R.P. J. Immunol. 1998; 161: 4366-4374PubMed Google Scholar). Since the total tyrosine phosphorylation of a molecule is the sum of the phosphorylation of such sites that have positive or negative functional effects, it is not surprising that strong tyrosine phosphorylation of Syk is not always a standard for downstream propagation of signals (37Swaim W.D. Minoguchi K. Oliver C. Hamawy M.M. Kihara H. Stephan V. Berenstein E.H. Siraganian R.P. J. Biol. Chem. 1994; 269: 19466-19473Abstract Full Text PDF PubMed Google Scholar). In the present experiments, we compared the FcεRI- and anti-ganglioside mAb AA4-induced histamine release to the total and activation loop tyrosine phosphorylation of Syk. We found that the phosphorylation of the activation loop tyrosines but not the total phosphorylation of Syk correlates with antigen- or AA4-mediated degranulation. Therefore, phosphorylation of the activation loop tyrosines rather than total phosphorylation is a better criterion for the functional activation of Syk. Although mAb AA4 induced minimal tyrosine phosphorylation of the activation loop, it resulted in tyrosine phosphorylation of many cellular proteins (37Swaim W.D. Minoguchi K. Oliver C. Hamawy M.M. Kihara H. Stephan V. Berenstein E.H. Siraganian R.P. J. Biol. Chem. 1994; 269: 19466-19473Abstract Full Text PDF PubMed Google Scholar). In contrast, there is minimal FcεRI-induced phosphorylation of cellular proteins in cells in which Syk activation loop tyrosines cannot be phosphorylated because they are mutated to Phe. There are several possible reasons for this difference in total cellular protein tyrosine phosphorylations. These mAb AA4-induced phosphorylations, unlike those by receptor aggregation, may not be due to Syk but my instead result from activation of other kinases such as Lyn. In fact, the ganglioside recognized by mAb AA4 is in membrane rafts and closely associated with Lyn (37Swaim W.D. Minoguchi K. Oliver C. Hamawy M.M. Kihara H. Stephan V. Berenstein E.H. Siraganian R.P. J. Biol. Chem. 1994; 269: 19466-19473Abstract Full Text PDF PubMed Google Scholar). It is also possible that the low level phosphorylation of the activation loop tyrosines in mAb AA4-stimulated cells may be enough to activate Syk to propagate downstream phosphorylation of proteins; however, this cannot occur in the activation loop mutated Syk. In both mAb AA4- and receptor-stimulated cells there was strong total phosphorylation of Syk, indicating that these stimuli result in differences in the ratio of the level of phosphorylation of tyrosines in the activation loop to other sites in the molecule. The ratio in the phosphorylation of different tyrosine residues that can either up- or down-regulate Syk function could then control downstream events. Both receptor aggregation and mAb AA4 induced strong tyrosine phosphorylation of Syk and other cellular proteins although only IgE-antigen resulted in degranulation. Total cellular tyrosine phosphorylations do not always correlate with degranulation. For example, when doubly negative CD45−/−Syk−/− cells are transfected with ZAP70 there is minimal tyrosine phosphorylation of cellular proteins without degranulation; the cotransfection with CD45 does not change this level of phosphorylation but results in degranulation (48Zhang J. Siraganian R.P. J. Immunol. 1999; 163: 2508-2516PubMed Google Scholar). Another difference is that the rate of the total tyrosine phosphorylation of Syk is much slower with mAb AA4 than with receptor activation (Fig. 2). This may be critical for the organization of the complex of molecules that are important for signaling. As discussed above, the ratio in the phosphorylation of different tyrosines that can either activate or inhibit Syk function could regulate downstream signals for degranulation. In the in vitro kinase reaction, there was autophosphorylation of Syk including phosphorylation of the activation loop tyrosines in the absence of any detectable Lyn. The binding of diphosphorylated ITAM results in a conformational change in Syk that stimulates kinase activity, thus increasing autophosphorylation (15Kimura T. Sakamoto H. Appella E. Siraganian R.P. Mol. Cell. Biol. 1996; 16: 1471-1478Crossref PubMed Scopus (101) Google Scholar) and the phosphorylation of the activation loop tyrosines (Fig. 4). Conformational changes in the molecule may therefore make activation loop tyrosines accessible for transphosphorylation. In vivo,there was low level phosphorylation of the activation loop tyrosines of the kinase-dead Syk, indicating the activity of another kinase, probably Lyn. These results suggest that Syk may be activated by several mechanisms that include conformational change due to ITAM binding, autophosphorylation, and phosphorylation by other kinases. In summary, a specific, anti-Syk activation loop phosphotyrosine antibody was generated. By using this antibody, we found that both of the tyrosines in the activation loop were phosphorylated following antigen stimulation. Syk played the major role in phosphorylating its activation loop tyrosines. In FcεRI-stimulated or anti-ganglioside mAb AA4-stimulated mast cells, only phosphorylation of the activation loop tyrosines, but not the total Syk tyrosine phosphorylation, correlated with the functional response of degranulation. Therefore, the tyrosine phosphorylation of the activation loop rather than total phosphorylation of Syk is a better indicator of in vivo Syk functional activation. We are grateful to Dr. Kiyonao Sada and Dr. Daniel Vial for reviewing the manuscript. We thank Lynda Weedon and Greta Bader for excellent technical help." @default.
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• W2005857763 title "Phosphorylation of Syk Activation Loop Tyrosines Is Essential for Syk Function" @default.
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