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• W2024891824 abstract "The Tec family tyrosine kinase, Itk has been implicated in T cell antigen receptor (TCR) signaling, yet little is known about Itk regulation. Here, we investigate the role of the tyrosine kinase ZAP-70 in regulating Itk. Whereas Itk was activated in Jurkat T cells in response to CD3 cross-linking, Itk activation was defective in the ZAP-70-deficient P116 Jurkat T cell line. Itk responsiveness to TCR engagement was restored in P116 cells stably transfected with ZAP-70 cDNA. ZAP-70 itself could not directly phosphorylate the Itk kinase domain, indicating an indirect regulation of Itk activity. No role was found for ZAP-70 in regulating Itk recruitment to the plasma membrane, an event that has been suggested to be rate-limiting for the activation of Tec family kinases. Indeed, Itk was found to be constitutively targeted to the membrane fraction in both Jurkat and P116 cells. Lat, a prominent in vivosubstrate of ZAP-70 that mediates assembly of multimolecular signaling complexes at the plasma membrane of T cells was also found to be required for TCR-stimulated Itk activation. Itk could not be activated by CD3 cross-linking in a Lat-negative cell line, unless Lat expression was restored. Lat and Itk were observed to co-associate in response to CD3 cross-linking in Jurkat T cells, but not in P116 T cells. The Lat-Itk association correlated with Lat tyrosine phosphorylation, which was deficient in the P116 T cells. These data suggest that ZAP-70 and Lat play important, probably sequential, roles in regulating the activation of Itk following TCR engagement. The Tec family tyrosine kinase, Itk has been implicated in T cell antigen receptor (TCR) signaling, yet little is known about Itk regulation. Here, we investigate the role of the tyrosine kinase ZAP-70 in regulating Itk. Whereas Itk was activated in Jurkat T cells in response to CD3 cross-linking, Itk activation was defective in the ZAP-70-deficient P116 Jurkat T cell line. Itk responsiveness to TCR engagement was restored in P116 cells stably transfected with ZAP-70 cDNA. ZAP-70 itself could not directly phosphorylate the Itk kinase domain, indicating an indirect regulation of Itk activity. No role was found for ZAP-70 in regulating Itk recruitment to the plasma membrane, an event that has been suggested to be rate-limiting for the activation of Tec family kinases. Indeed, Itk was found to be constitutively targeted to the membrane fraction in both Jurkat and P116 cells. Lat, a prominent in vivosubstrate of ZAP-70 that mediates assembly of multimolecular signaling complexes at the plasma membrane of T cells was also found to be required for TCR-stimulated Itk activation. Itk could not be activated by CD3 cross-linking in a Lat-negative cell line, unless Lat expression was restored. Lat and Itk were observed to co-associate in response to CD3 cross-linking in Jurkat T cells, but not in P116 T cells. The Lat-Itk association correlated with Lat tyrosine phosphorylation, which was deficient in the P116 T cells. These data suggest that ZAP-70 and Lat play important, probably sequential, roles in regulating the activation of Itk following TCR engagement. T cell antigen receptor interleukin-2-inducible T cell kinase Bruton's tyrosine kinase linker for activation of T cells phosphatidylinositol 3-kinase phospholipase Cγ glycolipid-enriched membrane protein tyrosine kinase glutathione S-transferase polyacrylamide gel electrophoresis kinase domain 4-morpholinepropanesulfonic acid Engagement of the T cell antigen receptor (TCR)1 initiates a complex cascade of biochemical events, which, in the context of co-stimulatory signals, culminate in proliferation and acquisition of effector functions by the T cell. The most receptor-proximal events initiated upon TCR stimulation are the activation of several protein tyrosine kinases (PTKs) of the Src, Syk, and Tec families (1Bolen J.B. Brugge J.S. Annu. Rev. Immunol. 1997; 15: 371-404Crossref PubMed Scopus (131) Google Scholar, 2Qian D. Weiss A. Curr. Opin. Cell Biol. 1997; 9: 205-212Crossref PubMed Scopus (286) Google Scholar). TCR signaling is initiated when the Src family PTKs Lck and Fyn phosphorylate specific tyrosine residues within the immunoreceptor tyrosine-based activation motifs of the CD3 (γ, δ, and ε) and TCRζ subunits of the TCR/CD3 complex (3Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar, 4van Oers N.S. Killeen N. Weiss A. J. Exp. Med. 1996; 183: 1053-1062Crossref PubMed Scopus (283) Google Scholar). The doubly phosphorylated immunoreceptor tyrosine-based activation motifs recruit the Syk family PTK ZAP-70 to the TCR via interaction with the tandem Src homology 2 domains of ZAP-70 (5Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar). Recruitment to the TCR is required for the subsequent tyrosine phosphorylation and activation of ZAP-70 (6Wange R.L. Isakov N. Burke Jr., T.R. Otaka A. Roller P.P. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 944-948Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 7Qian D. Mollenauer M.N. Weiss A. J. Exp. Med. 1996; 183: 611-620Crossref PubMed Scopus (93) Google Scholar). ZAP-70 lies at a key point in the TCR signaling pathway, being required for the activation of calcium mobilization and Erk activation pathways, which are in turn required for interleukin-2 production and T cell proliferation (7Qian D. Mollenauer M.N. Weiss A. J. Exp. Med. 1996; 183: 611-620Crossref PubMed Scopus (93) Google Scholar, 8Williams B.L. Schreiber K.L. Zhang W. Wange R.L. Samelson L.E. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (223) Google Scholar). This function is accomplished, in part, by regulating the formation of certain key multimolecular signaling complexes involved in these pathways, by catalyzing the phosphorylation of the hematopoietic-specific proteins SLP-76 and Lat (9Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 10Koretzky G.A. Immunol. Today. 1997; 18: 401-406Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 11Rudd C.E. Curr Biol. 1998; 8: R805-R808Abstract Full Text Full Text PDF PubMed Google Scholar). Both of these proteins have been shown to be in vivo substrates of ZAP-70 (12Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 13Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 14Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar). SLP-76, when tyrosine-phosphorylated, binds to another hematopoietic-specific protein, Vav, and has been implicated in playing an important, as yet undefined role in both Ca2+mobilization as well as Ras activation in T cells (12Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 15Wu J. Motto D.G. Koretzky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 16Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 281: 413-416Crossref PubMed Scopus (355) Google Scholar). Tyrosine-phosphorylated Lat, which is primarily resident in the glycolipid-enriched membrane (GEM) fraction of the plasma membrane, recruits PLCγ1, Grb2, and PI3-K to the GEMs (14Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar, 17Zhang W. Trible R.P. Samelson L.E. Immunity. 1998; 9: 239-246Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). The GEM membrane lipid rafts have been proposed to serve as platforms for signal transduction upon TCR engagement (18Montixi C. Langlet C. Bernard A.M. Thimonier J. Dubois C. Wurbel M.A. Chauvin J.P. Pierres M. He H.T. EMBO J. 1998; 17: 5334-5348Crossref PubMed Scopus (559) Google Scholar, 19Moran M. Miceli M.C. Immunity. 1998; 9: 787-796Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Localization of PLCγ1 and PI3-K to the GEMs positions these enzymes near their shared substrate, phosphatidylinositol 4,5-biphosphate, which is enriched in the GEM fraction, and may facilitate the tyrosine phosphorylation of PLCγ1. Grb2/SOS recruitment to the GEMs also serves to position SOS in the vicinity of its substrate, Ras, which is constitutively targeted to the GEMs (20Xavier R. Brennan T. Li Q. McCormack C. Seed B. Immunity. 1998; 8: 723-732Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). The Tec family tyrosine kinases have been implicated in antigen receptor signaling in a variety of hematopoietic cell types. Btk, a Tec family member primarily expressed in B cells and mast cells, is involved in B cell antigen receptor signaling and was found to be defective in the human and murine immunodeficiencies, X-linked agammaglobulinemia, and X-linked immunodeficiency, respectively (21Rawlings D.J. Saffran D.C. Tsukada S. Largaespada D.A. Grimaldi J.C. Cohen L. Mohr R.N. Bazan J.F. Howard M. Copeland N.G. Jenkins N.A. Witte O.N. Science. 1993; 261: 358-361Crossref PubMed Scopus (777) Google Scholar, 22Conley M.E. Rohrer J. Clin. Immunol. Immunopathol. 1995; 76: S192-S197Crossref PubMed Scopus (22) Google Scholar, 23Rawlings D.J. Witte O.N. Semin. Immunol. 1995; 7: 237-246Crossref PubMed Scopus (113) Google Scholar). Itk, also known as Emt or Tsk, is expressed in T cells and NK cells (24Siliciano J.D. Morrow T.A. Desiderio S.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11194-11198Crossref PubMed Scopus (235) Google Scholar, 25Gibson S. Leung B. Squire J.A. Hill M. Arima N. Goss P. Hogg D. Mills G.B. Blood. 1993; 82: 1561-1572Crossref PubMed Google Scholar, 26Tanaka N. Asao H. Ohtani K. Nakamura M. Sugamura K. FEBS Lett. 1993; 324: 1-5Crossref PubMed Scopus (39) Google Scholar); is tyrosine-phosphorylated in response to cross-linking of TCR, CD28, or CD2 (27August A. Gibson S. Kawakami Y. Kawakami T. Mills G.B. Dupont B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9347-9351Crossref PubMed Scopus (210) Google Scholar, 28Gibson S. August A. Kawakami Y. Kawakami T. Dupont B. Mills G.B. J. Immunol. 1996; 156: 2716-2722PubMed Google Scholar, 29Tanaka N. Abe H. Yagita H. Okumura K. Nakamura M. Sugamura K. Eur. J. Immunol. 1997; 27: 834-841PubMed Google Scholar); and has been implicated in thymocyte development and the activation of T cells through TCR and CD28 engagement. Mice engineered with a null mutation within the Itk gene have decreased numbers of mature thymocytes. Furthermore, T cells isolated from these mice are compromised in their proliferative response to allogeneic MHC stimulation, and to anti-TCR/CD3 cross-linking (30Liao X.C. Littman D.R. Immunity. 1995; 3: 757-769Abstract Full Text PDF PubMed Scopus (264) Google Scholar). These T cells also exhibit defective PLCγ1 tyrosine phosphorylation, inositol trisphosphate production, and Ca2+ influx in response to TCR cross-linking (31Liu K.Q. Bunnell S.C. Gurniak C.B. Berg L.J. J. Exp. Med. 1998; 187: 1721-1727Crossref PubMed Scopus (273) Google Scholar). How Itk activity is regulated in response to TCR engagement is still poorly understood. Structural studies have shown that Itk forms intramolecular interactions between its Src homology 3 domain and the proline-rich region of its Tec homology domain, and it has been proposed that these associations may be involved in regulating its activity (32Andreotti A.H. Bunnell S.C. Feng S. Berg L.J. Schreiber S.L. Nature. 1997; 385: 93-97Crossref PubMed Scopus (228) Google Scholar). Experiments in Jurkat T cells lacking Lck have demonstrated a requirement for Lck in Itk activation in response to TCR engagement (28Gibson S. August A. Kawakami Y. Kawakami T. Dupont B. Mills G.B. J. Immunol. 1996; 156: 2716-2722PubMed Google Scholar). Lck has also been shown to phosphorylate the critical activation loop tyrosine of Itk in vitro (33Heyeck S.D. Wilcox H.M. Bunnell S.C. Berg L.J. J. Biol. Chem. 1997; 272: 25401-25408Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The activation of Itk by G protein βγ subunits has also been reported (34Langhans-Rajasekaran S.A. Wan Y. Huang X.Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8601-8605Crossref PubMed Scopus (127) Google Scholar), but it remains unclear whether this plays any role in TCR-stimulated Itk activation. Experiments involving overexpression of Itk and c-Src in COS-7 cells demonstrated a requirement for PI3-K in recruiting Itk to the plasma membrane, where it became phosphorylated and activated by c-Src (35August A. Sadra A. Dupont B. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11227-11232Crossref PubMed Scopus (150) Google Scholar). Likewise, in A20 B cells, overexpression of the catalytic subunit of PI3-K was demonstrated to synergize with Btk, Itk, or Tec overexpression in stimulating IP3 production and the rise of [Ca2+]i in response to B cell antigen receptor cross-linking (36Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar). These latter studies by August et al. (35August A. Sadra A. Dupont B. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11227-11232Crossref PubMed Scopus (150) Google Scholar) and Scharenberget al. (36Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar) are consistent with the recently proposed model that Tec family kinases possessing pleckstrin homology domains (Itk/Tsk/Emt, Btk, and Tec) are regulated by changes in their subcellular localization. This model holds that receptor engagement activates PI3-K, which raises the plasma membrane concentration of phosphatidylinositol 3,4,5-trisphosphate, which is a high affinity binding site for the Tec family pleckstrin homology domain (37Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.-S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). This results in recruitment of Tec family kinases to the plasma membrane, where they are phosphorylated and activated by membrane-resident Src family kinases. Most of the evidence supporting this model has been gathered by study of Btk regulation (36Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar, 38Kawakami Y. Yao L. Miura T. Tsukada S. Witte O.N. Kawakami T. Mol. Cell. Biol. 1994; 14: 5108-5113Crossref PubMed Google Scholar, 39Li Z. Wahl M.I. Eguinoa A. Stephens L.R. Hawkins P.T. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13820-13825Crossref PubMed Scopus (187) Google Scholar, 40Bolland S. Pearse R.N. Kurosaki T. Ravetch J.V. Immunity. 1998; 8: 509-516Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Testing this model in Jurkat T cells, we found no evidence for TCR-stimulated Itk recruitment to the plasma membrane. Indeed, Itk was found to be constitutively present in the membrane fraction. In unstimulated Jurkat T cells, Itk could be recovered from both the GEM and bulk membrane fractions, and neither fraction exhibited any change in the Itk level upon TCR stimulation, arguing against redistribution of Itk to the plasma membrane as a mechanism for activation in Jurkat T cells. We also examined the role of ZAP-70 in Itk activation by measuring Itk activation in the ZAP-70-deficient P116 T cell line. We found that ZAP-70 is required for phosphorylation of Itk and activation of its kinase activity. However, ZAP-70 was unable to directly phosphorylate the kinase domain of Itk, suggesting that the role of ZAP-70 in regulating Itk tyrosine phosphorylation and activation is indirect. We also report the TCR-stimulated association of Itk with Lat, which was only observed in ZAP-70 replete cells. This association occurs with the same kinetics as Itk tyrosine phosphorylation and activation, and seems to be required for Itk activation in response to CD3 cross-linking, because OKT3-induced Itk activation was markedly reduced in Lat-deficient JCaM2.5 cells. The ZAP-70 deficient Jurkat T cell line, P116, has been previously described (8Williams B.L. Schreiber K.L. Zhang W. Wange R.L. Samelson L.E. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (223) Google Scholar, 41Griffith C.E. Zhang W. Wange R.L. J. Biol. Chem. 1998; 273: 10771-10776Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). P116 and the parental Jurkat E6 cells were the kind gift of R. Abraham (Duke University, Durham, NC). P.WT.18, a gift of L. Samelson (NCI, National Institutes of Health, Bethesda, MD), is a stable transfectant of P116 that expresses Myc-tagged, wild type ZAP-70 at a level comparable to the parental Jurkat line and has been characterized previously (41Griffith C.E. Zhang W. Wange R.L. J. Biol. Chem. 1998; 273: 10771-10776Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar,42Eischen C.M. Williams B.L. Zhang W. Samelson L.E. Lynch D.H. Abraham R.T. Leibson P.J. J. Immunol. 1997; 159: 1135-1139PubMed Google Scholar). The Lat-deficient Jurkat T cell mutant JCaM2.5 (43Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar) and its Lat-reconstituted cell line, JCaM2.5B3 2W. Zhang, R. Tribble, and L. E. Samelson, unpublished results. have been described and are the kind gifts of A. Weiss (University of California, San Francisco, CA) and L. Samelson (NCI, National Institutes of Health, Bethesda, MD). The JCaM2.5 cells show intact early signaling events, including tyrosine phosphorylation of CD3 chains and tyrosine phosphorylation and activation of ZAP-70, indicating that the Src family kinases Lck and Fyn function normally in these cells (43Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). All cells were maintained in RPMI 1640 medium supplemented with 7.5% fetal bovine serum (Hyclone), 10 μg/ml ciprofloxacin (Bayer), and 2 mm glutamine. The OKT3 monoclonal antibody to human CD3 and polyclonal rabbit antisera specific for human ZAP-70 and Lck have been described (6Wange R.L. Isakov N. Burke Jr., T.R. Otaka A. Roller P.P. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 944-948Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The anti-phosphotyrosine monoclonal antibody, 4G10 was from Upstate Biotechnology, Inc. (Lake Placid, NY). A polyclonal rabbit antiserum specific for human Itk was used to immunoprecipitate Itk and was kindly provided by G. Mills (University of Texas M. D. Anderson Cancer Center, Houston, TX). Itk was immunoblotted with the monoclonal antibody 2F12 directed against the N-terminal 26 amino acids of Itk, which was the gift of L. Berg (University of Massachusetts, Worcester, MA). The cytosolic domain of band III protein (cdb3) was prepared as described previously (44Wang C.C. Badylak J.A. Lux S.E. Moriyama R. Dixon J.E. Low P.S. Protein Sci. 1992; 1: 1206-1214Crossref PubMed Scopus (43) Google Scholar). GST fusion proteins containing the kinase domain of either Itk or Lck were the kind gift of J. Watts and R. Aebersold (University of Washington, Seattle, WA) and have been previously described (45Watts J.D. Brabb T. Bures E.J. Wange R.L. Samelson L.E. Aebersold R. FEBS Lett. 1996; 398: 217-222Crossref PubMed Scopus (10) Google Scholar). Cells were harvested by centrifugation, washed once, and resuspended in cold RPMI 1640 medium at a density of 1 × 108 cells/ml. After equilibration to 37 °C for 10 min, the cells were stimulated with OKT3 (1:50 ascites) for the indicated duration. Stimulation was terminated by addition of 5 volumes of 4 °C lysis buffer (20 mm Hepes (pH 7.4), 1% Triton X-100, 50 mm β-glycerophosphate, 2 mm EGTA, 10 mm sodium fluoride, 1 mm sodium orthovanadate, 10% glycerol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 100 μg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride). After a 30-min incubation on ice, postnuclear lysates were prepared by a 10-min centrifugation at 4 °C, 21,000 × g. The lysates were either directly analyzed by Western blotting or subjected to immunoprecipitation followed by immunoblotting or kinase assay. In some experiments, when indicated, a Brij 96 lysis buffer was used (1% Brij 96, 150 mm NaCl, 25 mm Tris-HCl (pH 7.5), 5 mm EDTA, 10 mm sodium fluoride, 1 mm sodium orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 100 μg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride). The postnuclear whole-cell lysates were incubated with protein A-agarose beads and corresponding antibodies for 2–16 h at 4 °C. In some of the anti-Itk immunoprecipitations, ZAP-70, Lck, and other TCR-associated proteins were depleted from the lysates by three rounds of OKT3 and/or anti-Lck immunoprecipitation. Immunoprecipitates that were to be analyzed by immunoblotting were washed three times with the above lysis buffer supplemented with 150 mm NaCl. Whole-cell lysates and immunoprecipitates to be analyzed by Western blotting were denatured by heating to 100 °C in Nu-PAGE sample buffer, electrophoresed on either 4–12% Nu-PAGE gradient or 6% Tris-glycine gels, and transferred to nitrocellulose membrane according to manufacturer's instructions (NOVEX, San Diego, CA). The blots were developed with the ECL system of Amersham Pharmacia Biotech and autoradiographed on BMR film (Eastman Kodak Co.). Itk-associated tyrosine kinase activity was assessed by immune complex kinase assay. Anti-Itk immunoprecipitates from lysates depleted of CD3 (and CD3-associated proteins) and Lck were washed twice with lysis buffer + 150 mm NaCl, twice with 4 °C LiCl wash buffer (100 mm Tris-HCl (pH 7.5), 0.5 m LiCl) and twice with 4 °C dH2O. To each sample of washed beads 30 μl of kinase reaction mixture (10 mm MgCl2, 10 mm Hepes (pH 7.0), 2 mm sodium orthovanadate, 5 μCi of [γ-32P]ATP, and 5 μg of RR-SRC substrate peptide (Sigma) were added. The reaction was performed at room temperature for 15 min with frequent mixing, then terminated by addition of acetic acid to 30% of the total volume. The reactions were centrifuged briefly and supernatants were spotted onto p81 phosphocellulose discs (Life Technologies, Inc.). After 4–6 washes with 75 mm phosphoric acid, the 32P incorporation was measured by liquid scintillation. In some assays, the kinase activity was normalized to the relative amount of Itk recovered in the anti-Itk immunoprecipitates. The relative amount of Itk was measured by densitometric analysis of x-ray films using the public domain NIH Image program (developed at the National Institutes of Health). The kinase reaction was carried out at 30 °C for 10 min in a total volume of 30 μl of reaction buffer (50 mmTris-HCl (pH 7.5), 10 mm MnCl2, 50 μm ATP, and 10 μCi of [γ-32P]ATP). The reaction was terminated by the addition of 10 μl of 4× reducing sample buffer and heating to 100 °C for 5 min. Phosphorylated proteins were resolved by SDS-PAGE and subjected to autoradiography. The GST fusion proteins containing the kinase domains of either Itk or Lck were purified from recombinant insect cells as described previously (45Watts J.D. Brabb T. Bures E.J. Wange R.L. Samelson L.E. Aebersold R. FEBS Lett. 1996; 398: 217-222Crossref PubMed Scopus (10) Google Scholar). The Itk-KD was cleaved away from the GST fusion partner by proteolytic cleavage with thrombin, and the GST fragment removed. Several of the preparations of Itk-KD prepared in this manner were found to have low intrinsic kinase activity, although they migrated normally on the gel. One of these low activity preparations was used as a substrate in the assay. The use of cdb3 as a ZAP-70 substrate has been described (6Wange R.L. Isakov N. Burke Jr., T.R. Otaka A. Roller P.P. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 944-948Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The ZAP-70 enzyme used in this assay was immunoprecipitated from activated (3 min at 37 °C with 1:100 OKT3) Jurkat whole-cell lysates that were depleted of Lck by three rounds of preclearing with antiserum recognizing Lck. The Lck enzyme used in the assay was the GST-Lck-KD fusion protein, which has been previously described (45Watts J.D. Brabb T. Bures E.J. Wange R.L. Samelson L.E. Aebersold R. FEBS Lett. 1996; 398: 217-222Crossref PubMed Scopus (10) Google Scholar). Cells (2.5 × 107) were centrifuged quickly in cold phosphate-buffered saline after OKT3 stimulation and resuspended in 1.5 ml of cold hypotonic lysis solution (20 mm Hepes (pH 7.6), 5 mm sodium pyrophosphate, 5 mm EGTA, 1 mm MgCl2, 10 μg/ml aprotinin, 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride), 1 mm sodium orthovanadate). The cell suspension sat on ice for 30 min, followed by cellular disruption with 10 passes of a Dounce homogenizer. After centrifugation at 100,000 × g at 4 °C for 1 h, the supernatant was collected as the cytosolic fraction. The pellet was rinsed with cold hypotonic lysis buffer and solubilized in 1.5 ml of the membrane solubilization solution (1% Triton X-100, 20 mm Hepes (pH 7.4), 150 mmNaCl, 1 mm MgCl2, 1 mm4-(2-aminoethyl)-benzenesulfonyl fluoride), 1 mm sodium orthovanadate) on ice for 30 min, followed by centrifugation at 100,000 × g at 4 °C for 1 h. This supernatant was taken as the membrane fraction. Cells (1 × 108) were centrifuged quickly in cold phosphate-buffered saline after OKT3 stimulation. The pellets were then lysed on ice in 1 ml of 1% Triton X-100 in TNEV buffer (10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 5 mm EDTA, 1 mmNa3VO4), with 15 strokes of a Dounce homogenizer, and mixed with 1 ml of 80% sucrose made with TNEV buffer. After transfer of the lysate to the centrifuge tube, 2 ml of 30% sucrose in TNEV buffer was overlaid, and then 1 ml of 5% sucrose in TNEV was overlaid. After centrifugation for 17 h at 200,000 × g in a Beckman SW55Ti, 0.4-ml gradient fractions were collected from the top of the gradient, in which the third fraction contained GEMs. To study the importance of ZAP-70 in Itk activation during TCR signaling, the human T cell line Jurkat and its ZAP-70-deficient mutant P116 were stimulated by CD3 cross-linking. As has been shown previously (28Gibson S. August A. Kawakami Y. Kawakami T. Dupont B. Mills G.B. J. Immunol. 1996; 156: 2716-2722PubMed Google Scholar, 33Heyeck S.D. Wilcox H.M. Bunnell S.C. Berg L.J. J. Biol. Chem. 1997; 272: 25401-25408Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) and in thetop panel of Fig.1 A, Itk was rapidly tyrosine-phosphorylated in Jurkat T cells upon OKT3 stimulation. Tyrosine phosphorylation could be detected within 45 s and returned to basal level by 15 min. In contrast, there was comparatively little increase in tyrosine phosphorylation of Itk in ZAP-70-negative cells receiving the same stimuli. The difference in Itk tyrosine phosphorylation in the two cell lines was not due to differences in Itk recovery, as the immunoprecipitates contained comparable amounts of Itk (Fig. 1 A, bottom panel). In the same experiment, we also examined whether or not ZAP-70 could regulate increased Itk kinase activity in response to TCR cross-linking. We compared the Itk kinase activity in both Jurkat and P116 cells, as measured by 32P incorporation into the substrate RR-SRC in an in vitro, immune-complex kinase assay (Fig. 1 B). OKT3 stimulation induced a rapid increase in the kinase activity recovered from Itk immunoprecipitates from Jurkat cells, but not in Itk immunoprecipitates from similarly treated ZAP-70-deficient P116 cells. Equal amounts of Itk were detected in the immunoprecipitates (Fig. 1 A, bottom panel). Although one group has reported that RR-SRC is not a good substrate for recombinant Itk (33Heyeck S.D. Wilcox H.M. Bunnell S.C. Berg L.J. J. Biol. Chem. 1997; 272: 25401-25408Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), others have reported the peptide to be a suitable substrate (28Gibson S. August A. Kawakami Y. Kawakami T. Dupont B. Mills G.B. J. Immunol. 1996; 156: 2716-2722PubMed Google Scholar), and we find RR-SRC to be a good substrate for both immunoprecipitated and recombinant (not shown) Itk. In particular, it is unlikely that the kinase activity measured in this assay is subject to interference from ZAP-70 or Lck, because RR-SRC is not a ZAP-70 substrate under the assay conditions used, and depletion of Lck from the lysates had no effect upon the OKT3-mediated activation of kinase activity recovered in Itk immunoprecipitations (not shown). To test whether the failure of tyrosine phosphorylation and subsequent kinase activation of Itk in P116 cells following TCR stimulation is due to the absence of ZAP-70, we examined the phosphorylation and activation of Itk in P.WT18 cells. This stable transfectant of P116 has been characterized previously, and expresses ZAP-70 at levels comparable to those observed in Jurkat (Fig.2 B, top panel, and Refs. 41Griffith C.E. Zhang W. Wange R.L. J. Biol. Chem. 1998; 273: 10771-10776Abstract Full Text Fu" @default.
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• W2024891824 title "Itk/Emt/Tsk Activation in Response to CD3 Cross-linking in Jurkat T Cells Requires ZAP-70 and Lat and Is Independent of Membrane Recruitment" @default.
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