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• W2000794723 abstract "Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates many of the biological activities of human neutrophils. The signaling pathways via which these effects are mediated are not fully understood. We have shown previously that GM-CSF treatment of human neutrophils activates the Janus kinase/signal transducers and activators of transcription (Jak/STAT) pathway and, more specifically, Jak2, STAT3, and STAT5B in neutrophils. GM-CSF also stimulates the activity of the phosphatidylinositol 3-kinase (PI3-kinase) in a tyrosine kinase-dependent manner. Here we report that pretreating the cells with a Jak2 inhibitor (AG-490) abolishes tyrosine phosphorylation of the p85 subunit of PI3-kinase induced by GM-CSF. Furthermore, p85 was found to associate with Jak2, but not with Lyn, in stimulated cells in situ and with its autophosphorylated form in vitro; however, Jak2 did not bind to either of the two Src homology 2 (SH2) domains of the p85 subunit of PI3-kinase. Although STAT5B bound to the carboxyl-terminal SH2 domain of p85, it was absent from the complex containing PI3-kinase and Jak2. These results suggest that stimulation of the activity of PI3-kinase induced by GM-CSF is mediated by Jak2 and that the association between Jak2 and p85 depends on an adaptor protein yet to be identified. Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates many of the biological activities of human neutrophils. The signaling pathways via which these effects are mediated are not fully understood. We have shown previously that GM-CSF treatment of human neutrophils activates the Janus kinase/signal transducers and activators of transcription (Jak/STAT) pathway and, more specifically, Jak2, STAT3, and STAT5B in neutrophils. GM-CSF also stimulates the activity of the phosphatidylinositol 3-kinase (PI3-kinase) in a tyrosine kinase-dependent manner. Here we report that pretreating the cells with a Jak2 inhibitor (AG-490) abolishes tyrosine phosphorylation of the p85 subunit of PI3-kinase induced by GM-CSF. Furthermore, p85 was found to associate with Jak2, but not with Lyn, in stimulated cells in situ and with its autophosphorylated form in vitro; however, Jak2 did not bind to either of the two Src homology 2 (SH2) domains of the p85 subunit of PI3-kinase. Although STAT5B bound to the carboxyl-terminal SH2 domain of p85, it was absent from the complex containing PI3-kinase and Jak2. These results suggest that stimulation of the activity of PI3-kinase induced by GM-CSF is mediated by Jak2 and that the association between Jak2 and p85 depends on an adaptor protein yet to be identified. granulocyte-macrophage colony-stimulating factor interleukin IL-1 receptor antagonist formylmethionylleucylphenylalanine Janus kinase signal transducers and activators of transcription phosphatidylinositol 3-kinase phosphatidylinositol src homology glutathione S-transferase Neutrophils are the most numerous of the white blood cells. They play, among others, a critical role in the nonspecific immune response. Several cytokines regulate the differentiation as well as the activity of neutrophils (1Smith J.A. J. Leukocyte Biol. 1994; 56: 672-686Crossref PubMed Scopus (754) Google Scholar, 2Hallett M.B. BioEssays. 1997; 19: 615-621Crossref PubMed Scopus (13) Google Scholar, 3Belova L.A. Biochemistry-Engl. Tr. 1997; 62: 563-570PubMed Google Scholar, 4Cassatella M.A. Immunol. Today. 1995; 16: 21-26Abstract Full Text PDF PubMed Scopus (827) Google Scholar). Among these is granulocyte-macrophage colony-stimulating factor (GM-CSF),1 a glycoprotein released by several cell types which supervises the maturation process of granulocyte and monocyte progenitors (5Gasson J.C. Weisbart R.H. Kaufman S.E. Clark S.C. Hewick R.M. Wong G.G. Golde D.W. Science. 1984; 226: 1339-1342Crossref PubMed Scopus (325) Google Scholar, 6Wiesbart R.H. Golde D.H. Clark S.C. Wong G.G. Gasson J.C. Nature. 1985; 314: 361-363Crossref PubMed Scopus (375) Google Scholar, 7Atkinson Y.H. Lopez A.F. Marasco W.A. Lucas C.M. Wong G.G. Burns G.F. Vadas M.A. Immunology. 1988; 64: 519-525PubMed Google Scholar). In addition, several of the biological activities of mature neutrophils are also regulated by GM-CSF, which acts mainly by priming these cells and making them more responsive to secondary stimuli. For example, superoxide production (7Atkinson Y.H. Lopez A.F. Marasco W.A. Lucas C.M. Wong G.G. Burns G.F. Vadas M.A. Immunology. 1988; 64: 519-525PubMed Google Scholar, 8Lopez A.F. Williamson D.J. Gamble J.R. Begley C.G. Harlan J.M. Klebanoff S.J. Waltersdorph A. Wong G. Clark S.C. Vadas M.A. J. Clin. Invest. 1986; 78: 1220-1228Crossref PubMed Scopus (541) Google Scholar, 9Nathan C.F. Blood. 1989; 73: 301-306Crossref PubMed Google Scholar) and phagocytosis (10Clark S. Int. J. Cell Cloning. 1988; 6: 365-377Crossref PubMed Scopus (31) Google Scholar) are both enhanced in neutrophils pretreated with GM-CSF as is the activation of several signaling pathways including the mobilization of calcium (11Naccache P.H. Faucher N. Borgeat P. Gasson J.C. DiPersio J.F. J. Immunol. 1988; 140: 3541-3546PubMed Google Scholar), the activation of phospholipase D (12Bourgoin S. Plante E. Gaudry M. Naccache P.H. Borgeat P. Poubelle P.E. J. Exp. Med. 1990; 172: 767-777Crossref PubMed Scopus (70) Google Scholar, 13Naccache P.H. Hamelin B. Gaudry M. Bourgoin S. Cell. Signalling. 1991; 3: 635-644Crossref PubMed Scopus (20) Google Scholar), and the stimulation of tyrosine phosphorylation (14Yuo A. Kitagawa S. Azuma E. Natori Y. Togawa A. Saito M. Takaku F. Biochim. Biophys. Acta. 1993; 1156: 197-203Crossref PubMed Scopus (53) Google Scholar, 15Kanakura Y. Druker B. Wood K.W. Mamon H.J. Okuda K. Roberts T.M. Griffin J.D. Blood. 1991; 77: 243-248Crossref PubMed Google Scholar, 16Roberts P.J. Khwaja A. Lie A.K.W. Bybee A. Yong K. Thomas N.S.B. Linch D.C. Blood. 1994; 84: 1064-1073Crossref PubMed Google Scholar). In addition, a number of direct effects of GM-CSF on neutrophils have been reported. These include the stimulated synthesis of interleukin (IL)-1 and IL-1 receptor antagonist (IL-1Ra) (10Clark S. Int. J. Cell Cloning. 1988; 6: 365-377Crossref PubMed Scopus (31) Google Scholar), increases in the surface expression of adhesion molecules of the β2 integrin family (17Sha'afi R.I. Molski T.F.P. Prog. Allergy. 1988; 42: 1-64PubMed Google Scholar), in the number as well as the affinity of fMLP receptors (7Atkinson Y.H. Lopez A.F. Marasco W.A. Lucas C.M. Wong G.G. Burns G.F. Vadas M.A. Immunology. 1988; 64: 519-525PubMed Google Scholar, 10Clark S. Int. J. Cell Cloning. 1988; 6: 365-377Crossref PubMed Scopus (31) Google Scholar), and induction of cytosolic alkalinization and tyrosine phosphorylation (14Yuo A. Kitagawa S. Azuma E. Natori Y. Togawa A. Saito M. Takaku F. Biochim. Biophys. Acta. 1993; 1156: 197-203Crossref PubMed Scopus (53) Google Scholar, 18Gomez-Cambronero J. Yamazaki M. Metwally F. Molski T.F.P. Bonak V.A. Huang C.K. Becker E.L. Sha'afi R.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3569-3573Crossref PubMed Scopus (64) Google Scholar). Although most of these activities have been well documented, the signaling mechanisms that underlay them are still poorly understood. The GM-CSF receptor is made up of two subunits, termed α and β (19Rapoport A.P. Abboud C.N. DiPersio J.F. Blood Rev. 1992; 6: 43-57Crossref PubMed Scopus (120) Google Scholar). The α subunit binds GM-CSF with low affinity, and only in presence of the β subunit is high affinity binding achieved (20Hayashida K. Kitamura T. Gorman D.M. Arai K.-I. Yokota T. Miyajima A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9655-9659Crossref PubMed Scopus (519) Google Scholar). Because of its short cytoplasmic tail, the α subunit was originally believed to play little role in signaling (19Rapoport A.P. Abboud C.N. DiPersio J.F. Blood Rev. 1992; 6: 43-57Crossref PubMed Scopus (120) Google Scholar, 21Polotskaya A. Zhao Y.M. Lilly M.L. Kraft A.S. Cell Growth Differ. 1993; 4: 523-531PubMed Google Scholar). This, however, is being challenged by recent reports suggesting its involvement in regulating cell growth (22Matsuguchi T. Zhao Y. Lilly M.B. Kraft A.S. J. Biol. Chem. 1997; 272: 17450-17459Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 23Muto A. Watanabe S. Itoh T. Miyajima A. Yokota T. Arai K. J. Allerg. Clin. Immunol. 1995; 96: 1100-1114Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The β subunit, on the other hand, has a long intracytoplasmic tail and plays a major role in transmitting the signal inside the cell (24Sakamaki K. Miyajima I. Kitamura T. Miyajima A. EMBO J. 1992; 11: 3541-3549Crossref PubMed Scopus (295) Google Scholar). Neither subunit has intrinsic enzymatic activity, nor are they known to link to G proteins directly (19Rapoport A.P. Abboud C.N. DiPersio J.F. Blood Rev. 1992; 6: 43-57Crossref PubMed Scopus (120) Google Scholar). Signaling by the GM-CSF receptor depends on the activation of the tyrosine phosphorylation pathways mediated by the stimulation of a number of cytosolic protein tyrosine kinases (25Quelle F.W. Sato N. Witthuhn B.S. Inhorn R.C. Eder M. Miyajima A. Griffin J.D. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4335-4341Crossref PubMed Google Scholar, 26Corey S. Eguinoa A. Puyanatheall K. Bolen J.B. Cantley L. Mollinedo F. Jackson T.R. Hawkins P.T. Stephens L.R. EMBO J. 1993; 12: 2681-2690Crossref PubMed Scopus (170) Google Scholar). In neutrophils, we and others have shown that three tyrosine kinases, Lyn (26Corey S. Eguinoa A. Puyanatheall K. Bolen J.B. Cantley L. Mollinedo F. Jackson T.R. Hawkins P.T. Stephens L.R. EMBO J. 1993; 12: 2681-2690Crossref PubMed Scopus (170) Google Scholar, 27Thompson N.T. Randall R.W. Garland L.G. Biochem. Soc. Trans. 1995; 23: S196Crossref Scopus (6) Google Scholar, 28Li Y. Shen B.F. Karanes C. Sensenbrenner L. Chen B. J. Immunol. 1995; 155: 2165-2174PubMed Google Scholar), Fes (29Brizzi M.F. Aronica M.G. Rosso A. Bagnara G.P. Yarden Y. Pegoraro L. J. Biol. Chem. 1996; 271: 3562-3567Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 30Hanazono Y. Chiba S. Sasaki K. Mano H. Miyajima A. Arai K. Yazaki Y. Hirai H. EMBO J. 1993; 12: 1641-1646Crossref PubMed Scopus (144) Google Scholar), and Jak2 (29Brizzi M.F. Aronica M.G. Rosso A. Bagnara G.P. Yarden Y. Pegoraro L. J. Biol. Chem. 1996; 271: 3562-3567Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 31Al-Shami A. Bourgoin S.G. Naccache P.H. Blood. 1997; 89: 1035-1044Crossref PubMed Google Scholar), are activated upon stimulation with GM-CSF. Each of these kinases is involved in the signaling pathways associated with various cytokines (25Quelle F.W. Sato N. Witthuhn B.S. Inhorn R.C. Eder M. Miyajima A. Griffin J.D. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4335-4341Crossref PubMed Google Scholar, 32Wojchowski D.M. He T.C. Stem Cells. 1993; 11: 381-392Crossref PubMed Scopus (32) Google Scholar, 33Witthuhn B.A. Silvennoinen O. Miura O. Lai K.S. Cwik C. Liu E.T. Ihle J.N. Nature. 1994; 370: 153-157Crossref PubMed Scopus (534) Google Scholar, 34Tanaka N. Asao H. Ohbo K. Ishii N. Takeshita T. Nakamura M. Sasaki H. Sugamura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7271-7275Crossref PubMed Scopus (65) Google Scholar). For example, Jak2 will associate with and phosphorylate the β subunit of the GM-CSF receptor upon ligand binding in TF-1 cells (25Quelle F.W. Sato N. Witthuhn B.S. Inhorn R.C. Eder M. Miyajima A. Griffin J.D. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4335-4341Crossref PubMed Google Scholar). The Jak family functions upstream of a family of transcription factors called STATs (signal transducers and activators of transcription) (35Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (820) Google Scholar,36Ihle J.N. Philos. Trans. R. Soc. Lond.-Biol. Sci. 1996; 351: 159-166Crossref PubMed Scopus (66) Google Scholar). Eight different STATs have been identified so far (STAT1 α and β, STAT2–4, STAT5A and B, and STAT6). The STATs are activated by tyrosine phosphorylation (37Pellegrini S. Dusanter-Fourt I. Eur. J. Biochem. 1997; 248: 615-633Crossref PubMed Scopus (237) Google Scholar) and in some cases by an additional serine phosphorylation (38Wen Z. Zhong Z. Darnell J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1735) Google Scholar). The phosphorylated STATs will form homo- as well as heterodimers and migrate to the nucleus where they bind specific DNA motifs (35Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (820) Google Scholar, 36Ihle J.N. Philos. Trans. R. Soc. Lond.-Biol. Sci. 1996; 351: 159-166Crossref PubMed Scopus (66) Google Scholar, 39Horvath C.M. Darnell J.E. Curr. Opin. Cell Biol. 1997; 9: 233-239Crossref PubMed Scopus (175) Google Scholar). Which members of the Jak and STAT family are activated varies greatly among different agonists and for the same agonist but in different cell systems (29Brizzi M.F. Aronica M.G. Rosso A. Bagnara G.P. Yarden Y. Pegoraro L. J. Biol. Chem. 1996; 271: 3562-3567Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 40Mui A.L.F. Wakao H. O'Farrell A.M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (537) Google Scholar, 41Rosen R.L. Winestock K.D. Chen G. Liu X.W. Hennighausen L. Finbloom D.S. Blood. 1996; 88: 1206-1214Crossref PubMed Google Scholar, 42Dandrea R. Rayner J. Moretti P. Lopez A. Goodall G.J. Gonda T.J. Vadas M. Blood. 1994; 83: 2802-2808Crossref PubMed Google Scholar, 43Dorsch M. Hock H. Diamantstein T. Biochem. Biophys. Res. Commun. 1994; 200: 562-568Crossref PubMed Scopus (21) Google Scholar). In neutrophils, we have shown that GM-CSF treatment induces the selective activation of Jak2, STAT3, and STAT5B (44Al-Shami A. Mahana W. Naccache P.H. J. Biol. Chem. 1998; 273: 1058-1063Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Another effector system activated by GM-CSF involves phosphatidylinositol 3-kinase (PI3-kinase) (26Corey S. Eguinoa A. Puyanatheall K. Bolen J.B. Cantley L. Mollinedo F. Jackson T.R. Hawkins P.T. Stephens L.R. EMBO J. 1993; 12: 2681-2690Crossref PubMed Scopus (170) Google Scholar). This enzyme phosphorylates the third hydroxyl group of the inositol ring. In vitro, it uses phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns-4-P), and phosphatidylinositol 4,5-diphosphate (PtdIns-4,5-P2) as substrates leading to the production of PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3, respectively (45Liscovitch M. Cantley L.C. Cell. 1994; 77: 329-334Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 46Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N.F. Hsuan J.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (540) Google Scholar, 47Zhang X.L. Majerus P.W. Semin. Cell. Dev. Biol. 1998; 9: 153-160Crossref PubMed Scopus (59) Google Scholar). In vivo, however, only the levels of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are modulated by PI3-kinase upon stimulation, whereas the levels of PtdIns-3-P remain unchanged (47Zhang X.L. Majerus P.W. Semin. Cell. Dev. Biol. 1998; 9: 153-160Crossref PubMed Scopus (59) Google Scholar). PI3-kinase has been implicated in regulating cell proliferation (48Powis G. Phil D. Cancer Metastasis Rev. 1994; 13: 91-103Crossref PubMed Scopus (25) Google Scholar), protein secretion (49Yano H. Nakanishi S. Kimura K. Hanai N. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. J. Biol. Chem. 1993; 268: 25846-25856Abstract Full Text PDF PubMed Google Scholar), and membrane ruffling (50Wennstrom S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar) in various cell systems. In addition, the products catalyzed by the activation of PI3-kinase, namely PtdIns-3,4-P2 and PtdIns-3,4,5-P3, serve as cofactors for several members of the calcium-insensitive protein kinase C family (51Toker A. Meyer M. Reddy K.K. Falck J.R. Aneja R. Aneja S. Parra A. Burns D.J. Ballas L.M. Cantley L.C. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar, 52Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar) as well as other enzymes such as protein kinase B (53Marte B.M. Downward J. Trends Biochem. Sci. 1997; 22: 355-358Abstract Full Text PDF PubMed Scopus (644) Google Scholar, 54Stokoe D. Stephens L.R. Copeland T. Gaffney P.R.J. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1045) Google Scholar, 55Kennedy S.G. Wagner A.J. Conzen S.D. Jordan J. Bellacosa A. Tsichlis P.N. Hay N. Gene Dev. 1997; 11: 701-713Crossref PubMed Scopus (979) Google Scholar). Several members of the PI3-kinase family have been identified so far. The α, β, and δ are made up of a dimer containing a p85 regulatory subunit and a p110 catalytic subunit (56Domin J. Waterfield M.D. FEBS Lett. 1997; 410: 91-95Crossref PubMed Scopus (209) Google Scholar, 57Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (829) Google Scholar, 58Vanhaesebroeck B. Welham M.J. Kotani K. Stein R. Warne P.H. Zvelebil M.J. Higashi K. Volinia S. Downward J. Waterfield M.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4330-4335Crossref PubMed Scopus (370) Google Scholar). PI3-kinase γ is made up of a p110 catalytic subunit and a p101 regulatory subunit apparently activated by G-protein-linked receptors (31Al-Shami A. Bourgoin S.G. Naccache P.H. Blood. 1997; 89: 1035-1044Crossref PubMed Google Scholar, 59Stephens L.R. Eguinoa A. Erdjumentbromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). We have shown that GM-CSF treatment significantly activated PI3-kinase α in human neutrophils and that this activation is mediated by tyrosine kinases (31Al-Shami A. Bourgoin S.G. Naccache P.H. Blood. 1997; 89: 1035-1044Crossref PubMed Google Scholar). However, the identity of the kinases involved remains elusive. The aim of this report was to examine the potential interrelationships between the Jak/STAT and PI3-kinase pathways during the stimulation of human neutrophils by GM-CSF. The results presented show that inhibition of the tyrosine kinase activity of Jak2 abolished the GM-CSF-induced tyrosine phosphorylation of the p85 subunit of PI3-kinase associated with it. In addition, the p85 subunit was found to associate with Jak2 upon stimulation with GM-CSF in situ as well as with autophosphorylated Jak2 in vitro. Finally, STAT5B but not STAT3 associated with the carboxyl SH2 domain of p85 in cellular lysates of cells treated with GM-CSF. GM-CSF was generously provided by the Genetics Institute (Cambridge, MA). Nonidet P-40 was obtained from Sigma. Sephadex G-10, protein A, dextran T-500, and Ficoll-Paque were purchased from Amersham Pharmacia Biotech. The monoclonal antiphosphotyrosine antibody UB 05-321, the agarose-conjugated antiphosphotyrosine antibodies, the polyclonal anti-Jak2 (06-255) and anti-PI3-kinase (06-195) antibodies as well as purified Jak2 (14-134) were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Polyclonal antibodies to Lyn (sc-15), STAT3 (sc-482), and STAT5B (sc-835) were obtained from Santa Cruz Biotechnology Inc (Santa Cruz, Ca). The GST fusion proteins carrying the SH2 domains of the p85 subunit of PI3-kinase were a generous gift from Dr. Tony Pawson (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada). The enhanced chemiluminescence (ECL) Western blotting system was obtained from Amersham Pharmacia Biotech. Blood was collected from healthy adult volunteers into heparinized tubes. The cells were centrifuged for 10 min at 1,000 rpm to remove platelet-rich plasma. After 2% dextran sedimentation of erythrocytes for 30 min, neutrophils were purified under sterile conditions by centrifugation on Ficoll-Paque cushions. Contaminating erythrocytes were removed by hypotonic lysis, and the cells were suspended in magnesium-free Hanks' balanced salt solution containing 1.6 mm CaCl2 at a final count of 40 × 106 cells/ml. The entire procedure was carried out in sterile conditions at room temperature. The final cell preparation comprised at least 97% neutrophils and less than 0.2% monocytes. Neutrophil suspensions (1 ml of 40 × 106 cells/ml) were either stimulated with 4 nm GM-CSF or treated with the same volume of the diluent (0.01% bovine serum albumin) for the indicated periods of time at 37 °C. For lysates prepared under reducing conditions (60Al-Shami A. Gilbert C. Barabé F. Gaudry M. Naccache P.H. J. Immunol. Methods. 1997; 202: 183-191Crossref PubMed Scopus (20) Google Scholar), 500 μl of the cell suspensions was added to equal amounts of denaturing buffer A containing 50 mm Tris-HCl, pH 8.0, 150 mmNaCl, 2 mm EDTA, 50 mm NaF, 2 mmNaVO4, 20 mm NaP2O4, 10 μg/ml leupeptin,10 μg/ml aprotinin, 1 μm pepstatin, 1 mm phenylmethylsulfonyl fluoride, 1% SDS, and 0.6% β-mercaptoethanol (final concentrations) preheated to 100 °C, and incubated for 10 min. The lysates were centrifuged at 12,000 rpm for 10 min at room temperature. The supernatants were then filtered through Sephadex G-10 columns to remove the denaturing agents. To prepare the columns, 3 g of Sephadex/sample was suspended in 10 ml of buffer containing 20 mm Tris-HCl, pH 8.0, 5 mm EDTA, 5 mm EGTA, and 137 mm NaCl (final concentrations) for at least 3 h at room temperature with occasional shaking before use. Nonidet P-40 (1%) and bovine serum albumin (0.1 mg/ml) were added to the eluates which were subsequently used for immunoprecipitation. Lysates (1 ml) obtained as described above were incubated with 10 μg of agarose-conjugated antiphosphotyrosine antibodies or agarose-conjugated SH2 domains of the p85 subunit of PI3-kinase for 5 h at 4 °C with constant rotation. The beads were collected by centrifugation and washed twice with modified buffer A containing 1% Nonidet P-40 but no SDS or β-mercaptoethanol and twice with LiCl buffer (0.5 m LiCl, 20 mm Hepes, pH 7.4). The supernatants were removed carefully, 45 μl of 2 × boiling Laemmli buffer (1 × is 62.5 mTris-HCl, pH 6.8, 4% SDS, 5% β-mercaptoethanol, 8.5% glycerol, 2.5 mm NaVO4, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 0.025% bromphenol blue) was then added, and the samples were boiled for 10 min before being used for electrophoresis. For nondenaturing lysis, buffers containing 20 mm Tris-HCl, pH 7.4, 137 mm NaCl, 10% glycerol, 25 mm NaF, 2 mm NaVO4, 1 μm pepstatin, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mmphenylmethylsulfonyl fluoride, and 1% Nonidet P-40 (final concentrations) were used. The lysed cells were incubated on ice for 10 min before centrifugation at 12,000 rpm for 20 min. A preclearing with protein A-Sepharose for 30 min at 4 °C was carried out. The lysates were transferred carefully to new tubes and used for subsequent immunoprecipitation with anti-Jak2 and anti-Lyn antibodies for 2 h at 4 °C. 20 μg of protein A-Sepharose was added next, and the tubes were left at 4 °C for 1 h. The beads were then washed twice with lysis buffer and twice with LiCl buffer (0.5 mLiCl, 20 mm Hepes, pH 7.4). Agarose-conjugated purified Jak2 was suspended in kinase buffer containing 20 mm Hepes, pH 7.4, 1 mmMgCl2, 1 mm MnCl2, 25 mm NaCl, and 0.1 mm NaVO4 for 40 min at 30 °C with or without 1 mm ATP. The enzyme (10 μg) was washed with phosphate-buffered saline and incubated with 1 ml of cellular lysates taken from untreated neutrophils that were prepared under native conditions as described above. The mixture was left at 4 °C for 2 h before being washed twice with LiCl buffer (0.5m LiCl, 20 mm Hepes, pH 7.4). Neutrophils (4 × 106 cells/ml) were treated with 200 μm AG-490 or diluent (dimethyl sulfoxide) for 90 min at 37 °C. The assay to measure the activity of PI3-kinase was conducted as described previously (61Gold M.R. Duronio V. Saxena S.P. Schrader J.W. Aebersold R. J. Biol. Chem. 1994; 269: 5403-5412Abstract Full Text PDF PubMed Google Scholar). Briefly, PI3-kinase was immunoprecipitated by adding 4 μg of anti-p85 antibodies to 900 μl of nondenatured precleared lysates and left at 4 °C for 2 h. The antibodies were collected after a 1-h incubation at 4 °C with protein A-Sepharose. The immunoprecipitates were washed once with each of the following: lysis buffer, LiCl buffer (0.5 m LiCl, 100 mm Tris-HCl, pH 7.4), EDTA buffer (1 mmEDTA, 100 mm NaCl, 10 mm Tris-HCl, pH 7.4), and kinase buffer without ATP (20 mm Tris-HCl, pH 7.5, 5 mm MnCl2, 5 mm MgCl2). The immunoprecipitates were suspended in 50 μl of kinase buffer containing 300 μg/ml phosphatidylinositol substrate and 10 μCi of [32P]ATP. The reactions were allowed to proceed for 90 s before being quenched with 80 μl of 1 n HCl. The lipids were extracted by the addition of 200 μl of chloroform/methanol (1:1 v/v ratio) and separated on thin layer chromatography plates precoated with ammonium oxalate. The migration solution contained 2-propanol and 2 m acetic acid at a 2:1 v/v ratio. The phosphorylated lipids were revealed by autoradiography. The specificity of PI3-kinase activity was confirmed by its inhibition in the presence of either 20 nm wortmannin or 2.5% Nonidet P-40. Samples were electrophoresed on 7.5–20% SDS-polyacrylamide gradient gels. Electrophoretic transfer cells (Hoefer Scientific Instruments, Canberra Packard, Ontario, Canada) were used to transfer proteins from the gels to Immobilon polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA). Immunoblotting was performed as described previously (34Tanaka N. Asao H. Ohbo K. Ishii N. Takeshita T. Nakamura M. Sasaki H. Sugamura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7271-7275Crossref PubMed Scopus (65) Google Scholar). Briefly, nonspecific sites were blocked using 2% gelatin in TBS-Tween (25 mm Tris-HCl, pH 7.8, 190 mm NaCl, 0.15% Tween 20) for 1 h at 37 °C. The first immunoblot was carried out with the indicated antibodies that were incubated with the membranes for 1 h at 37 °C at a final dilution of 1/4,000 in fresh blocking solution. The membranes were washed three times at room temperature in TBS-Tween for a total duration of 30 min and then incubated with horseradish peroxidase-labeled secondary antibodies for 1 h at 37 °C at a final dilution of 1/20,000 in fresh blocking solution. The membranes were washed three times with TBS-Tween, and the protein bands were revealed using the ECL Western blotting detection system following the manufacturer's directions. To ensure the presence of equal amounts of immunoprecipitated proteins under each condition, the membranes were routinely reblotted with the respective immunoprecipitating antibody. Reprobing was conducted as follows. The polyvinylidene difluoride membranes were treated with TBS buffer containing 1% H2O2 for 5 min at room temperature. The membranes were washed extensively with TBS buffer containing no H2O2 and were then blotted with the immunoprecipitating antibodies as described above. The first experiment was designed to examine the effect of AG-490, a reportedly specific Jak2 inhibitor (62Meydan N. Grunberger T. Dadi H. Shahar M. Arpaia E. Lapidot Z. Steven Leeder J. Freedman M. Cohen A. Gazit A. Levitzki A. Roifman C.H. Nature. 1996; 379: 645-648Crossref PubMed Scopus (847) Google Scholar), on the pattern of tyrosine phosphorylation of cellular proteins induced by GM-CSF in human neutrophils. Cells at 4 × 106/ml were incubated with 200 μm AG-490 or its diluent (dimethyl sulfoxide) for 90 min at 37 °C. The cells were then suspended in Hanks' balanced salt solution at 40 × 106/ml and stimulated with 4 nm GM-CSF or diluent (0.01% BSA) for 10 min at 37 °C and subsequently added to an equal volume of boiling sample buffer. Cellular proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted with antiphosphotyrosine antibodies. As shown in Fig.1, GM-CSF induced tyrosine phosphorylation of a number of proteins in the 150–140-, 120-, 90-, 80-, 70-, 55-, and 40-kDa region (63Kanakura Y. Druker B. DiCarlo J. Cannistra S.A. Griffin J.D. J. Biol. Chem. 1991; 266: 490-495Abstract Full Text PDF PubMed Google Scholar, 64McColl S.R. DiPersio J.F. Caon A.C. Ho P. Naccache P.H. Blood. 1991; 78: 1842-1852Crossref PubMed Google Scholar). Treating cells with AG-490 led to a decrease in the level of tyrosine phosphorylation of most of these proteins especially those with molecular masses in the 140-, 120-, 90-, 80-, and 40-kDa ranges (indicated by arrows). AG-490 was used at 200 μm, a concentration that provided the optimal inhibition of Jak2 within 90 min of preincubation without affecting the survival of the cells as evidenced from testing by trypan blue (data not shown). In addition, neutrophils pretreated with AG-490 were responsive to treatment with IL-8 and fMLP, two agonists that utilize G-protein-linked receptors (data not shown). The inhibitory effects of AG-490 were time- and concentration-dependent. Shorter incubation times (30–60 min) or lower concentrations (50, 100, 150 μm) inhibited progressively less the tyrosine phosphorylation stimulated by GM-CSF (data not shown). We have shown previously that the stimulation of human neutrophils by GM-CSF induced tyrosine phosphorylation of the p85 subunit and activation of PI3-kinase (31Al-Shami A. Bourgoin S.G. Naccache P.H. Blood. 1997; 89: 1035-1044Crossref PubMed Google Scholar). We examined next the effect of AG-490 on these two parameters. Cells were incubated with AG-490 as described above and treated with 4 nm GM-CSF or diluent for 15 min at 37 °C. The" @default.
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• W2000794723 title "Granulocyte-Macrophage Colony-stimulating Factor-activated Signaling Pathways in Human Neutrophils" @default.
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