The CC Chemokine Monocyte Chemotactic Peptide-1 Activates both the Class I p85/p110 Phosphatidylinositol 3-Kinase and the Class II PI3K-C2α
1998; Elsevier BV; Volume: 273; Issue: 40 Linguagem: Inglês
10.1074/jbc.273.40.25987
ISSN1083-351X
AutoresSarah J. Turner, Jan Domin, Michael D. Waterfield, Stephen G. Ward, John Westwick,
Tópico(s)interferon and immune responses
ResumoThe cellular effects of MCP-1 are mediated primarily by binding to CC chemokine receptor-2. We report here that MCP-1 stimulates the formation of the lipid products of phosphatidylinositol (PI) 3-kinase, namely phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3) in THP-1 cells that can be inhibited by pertussis toxin but not wortmannin. MCP-1 also stimulates an increase in the in vitro lipid kinase activity present in immunoprecipitates of the class 1A p85/p110 heterodimeric PI 3-kinase, although the kinetics of activation were much slower than observed for the accumulation of PI 3,4,5-P3. In addition, this in vitro lipid kinase activity was inhibited by wortmannin (IC50 = 4.47 ± 1.88 nm,n = 4), and comparable concentrations of wortmannin also inhibited MCP-stimulated chemotaxis of THP-1 cells (IC50 = 11.8 ± 4.2 nm, n= 4), indicating that p85/p110 PI 3-kinase activity is functionally relevant. MCP-1 also induced tyrosine phosphorylation of three proteins in these cells, and a fourth tyrosine-phosphorylated protein co-precipitates with the p85 subunit upon MCP-1 stimulation. In addition, MCP-1 stimulated lipid kinase activity present in immunoprecipitates of a class II PI 3-kinase (PI3K-C2α) with kinetics that closely resembled the accumulation of PI 3,4,5-P3. Moreover, this MCP-1-induced increase in PI3K-C2α activity was insensitive to wortmannin but was inhibited by pertussis toxin pretreatment. Since this mirrored the effects of these inhibitors on MCP-1-stimulated increases in D-3 phosphatidylinositol lipid accumulation in vivo, these results suggest that activation of PI3K-C2α rather than the p85/p110 heterodimer is responsible for mediating the in vivo formation of D-3 phosphatidylinositol lipids. These data demonstrate that MCP-1 stimulates protein tyrosine kinases as well as at least two separate PI 3-kinase isoforms, namely the p85/p110 PI 3-kinase and PI3K-C2α. This is the first demonstration that MCP-1 can stimulate PI 3-kinase activation and is also the first indication of an agonist-induced activation of the PI3K-C2α enzyme. These two events may play important roles in MCP-1-stimulated signal transduction and biological consequences. The cellular effects of MCP-1 are mediated primarily by binding to CC chemokine receptor-2. We report here that MCP-1 stimulates the formation of the lipid products of phosphatidylinositol (PI) 3-kinase, namely phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3) in THP-1 cells that can be inhibited by pertussis toxin but not wortmannin. MCP-1 also stimulates an increase in the in vitro lipid kinase activity present in immunoprecipitates of the class 1A p85/p110 heterodimeric PI 3-kinase, although the kinetics of activation were much slower than observed for the accumulation of PI 3,4,5-P3. In addition, this in vitro lipid kinase activity was inhibited by wortmannin (IC50 = 4.47 ± 1.88 nm,n = 4), and comparable concentrations of wortmannin also inhibited MCP-stimulated chemotaxis of THP-1 cells (IC50 = 11.8 ± 4.2 nm, n= 4), indicating that p85/p110 PI 3-kinase activity is functionally relevant. MCP-1 also induced tyrosine phosphorylation of three proteins in these cells, and a fourth tyrosine-phosphorylated protein co-precipitates with the p85 subunit upon MCP-1 stimulation. In addition, MCP-1 stimulated lipid kinase activity present in immunoprecipitates of a class II PI 3-kinase (PI3K-C2α) with kinetics that closely resembled the accumulation of PI 3,4,5-P3. Moreover, this MCP-1-induced increase in PI3K-C2α activity was insensitive to wortmannin but was inhibited by pertussis toxin pretreatment. Since this mirrored the effects of these inhibitors on MCP-1-stimulated increases in D-3 phosphatidylinositol lipid accumulation in vivo, these results suggest that activation of PI3K-C2α rather than the p85/p110 heterodimer is responsible for mediating the in vivo formation of D-3 phosphatidylinositol lipids. These data demonstrate that MCP-1 stimulates protein tyrosine kinases as well as at least two separate PI 3-kinase isoforms, namely the p85/p110 PI 3-kinase and PI3K-C2α. This is the first demonstration that MCP-1 can stimulate PI 3-kinase activation and is also the first indication of an agonist-induced activation of the PI3K-C2α enzyme. These two events may play important roles in MCP-1-stimulated signal transduction and biological consequences. monocyte chemotactic peptide-1 high performance liquid chromatography monoclonal antibody phosphatidylinositol phosphatidylinositol 3-monophosphate 4-P2, phosphatidylinositol 3,4-bisphosphate 4,5-P3, phosphatidylinositol 3,4,5-trisphosphate protein-tyrosine kinase regulated on activation normal T cell expressed and secreted CC chemokine receptor 2. Chemokines are a rapidly growing superfamily of 8–10-kDa peptides that selectively attract and activate leukocyte populations (1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar, 2Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar). Monocyte chemotactic peptide-1 (MCP-1)1 (1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar) is a member of the CC chemokine family (1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar, 2Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar), is a potent inducer of monocyte and CD45RO+ lymphocyte chemotaxis (3Furutani Y. Nomura M. Notake Y. Oyamada T. Fukui N. Yamada C.G. Oppenheim J.J. Matsushima K. Biochem. Biophys Res. Commun. 1989; 159: 249-255Crossref PubMed Scopus (220) Google Scholar, 4Carr M.W. Roth S.J. Luther E. Rose S.S. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3652-3656Crossref PubMed Scopus (1049) Google Scholar), and also activates host defense mechanisms such as superoxide release (5Zachariae C.O.C. Anderson A.O. Thompson H.L. Appella E. Mantovani A. Oppenheim J.J. Matsushima K. J. Exp. Med. 1990; 171: 2177-2182Crossref PubMed Scopus (202) Google Scholar). In vivo studies suggest that MCP-1 recruits monocytes to sites of inflammation in a variety of pathological conditions including atherosclerosis (6Nelken N.A. Coughlin S.R. Gordon D. Wilcox J.N. J. Clin. Invest. 1991; 90: 772-779Google Scholar) and rheumatoid arthritis (7Koch A.E. Kunkel S.L. Harlow L.A. Johnson B. Evanoff H.L. Haines G.K. Burdick M.D. Pope R.M. Streiter R.M. J. Clin. Invest. 1992; 90: 772-779Crossref PubMed Scopus (590) Google Scholar) as well as pulmonary fibrosis and granulomatous lung disease (8Chensue S.W. Warmington K.S. Ruth J.H. Sanghi P.S. Lincoln P. Kunkel S.L. J. Immunol. 1996; 157: 4602-4608PubMed Google Scholar). MCP-1 has also been demonstrated to augment cytotoxic lymphocyte and natural killer cell activity in vitro, suggesting a novel role for chemokines as costimulators of T cell activation (9Taub D.D. Ortaldo J.R. Turcovski-Corrales S.M. Key M.L. Longo D.L. Murphy W.J. J. Leukocyte Biol. 1996; 59: 81-89Crossref PubMed Scopus (202) Google Scholar). Support for MCP-1's importance in the physiology of inflammation comes from demonstrations in transgenic mice that it functions as a monocyte chemoattractantin vivo (10Fuentes M.E. Durham S.K. Swerdel M.R. Lewin A.C. Barton D.S. Megill J.R. Bravo R. Lira S.A. J. Immunol. 1995; 155: 5769-5776PubMed Google Scholar, 11Grewal I.S. Rutledge B.J. Fiorillo J.A. Gu L. Gladue R.P. Flavell R.A. Rollins B.J. J. Immunol. 1997; 159: 401-408PubMed Google Scholar, 12Gunn M.D. Nelken N.A. Liao X. Williams L.T. J. Immunol. 1997; 159: 401-408PubMed Google Scholar). Moreover, abnormalities in monocyte recruitment and cytokine expression are observed in MCP-1-deficient mice (13Lu B. Rutledge B.J. Gu L. Fiorillo J. Lukacs N.W. Kunkel S.L. North R. Gerard C. Rollins B.J. J. Exp. Med. 1998; 187: 601-608Crossref PubMed Scopus (889) Google Scholar). The effects of chemokines are mediated by a family of closely related G protein-coupled receptors (1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar). MCP-1 binds to CC chemokine receptor 2 (CCR2), which exists in A or B forms that arise via alternative splicing of the carboxyl-terminal tail (14Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2752-2756Crossref PubMed Scopus (653) Google Scholar). CCR2 is activated by multiple agonists, including MCP-2 (15Gong X. Gong W. Kuhns D.B. Ben-Baruch A. Howard O.M.Z. Wang J.M. J. Biol. Chem. 1997; 272: 11682-11685Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), MCP-3 (16Franci C. Wong L.M. Van Damme J. Proost P. Charo I.F. J. Immunol. 1995; 154: 6511-6517PubMed Google Scholar, 17Combadiere C. Ahuja S.K. Van Damme J. Tiffany H.L. Gao J. Murphy P.M. J. Biol. Chem. 1995; 270: 29671-29675Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), MCP-4 (18Garcia-Zepeda E.A. Combadiere C. Rothenberg M.E. Sarafi M.N. Lavigne F. Hamid Q. Murphy P.M. Luster A.D. J. Immunol. 1996; 157: 5613-5626PubMed Google Scholar,19Stellato C.P. Collins P. Ponath P.D. Soler D. Newman W. La Rosa G. Li H. White J. Schwiebert L.M. Bickel C. Liu M. Bochner B.S. Williams T.J. Schleimer R.P. J. Clin. Invest. 1997; 99: 926-936Crossref PubMed Scopus (170) Google Scholar), and MCP-5 (20Sarafi M.N. Garcia-Zepeda E.A. Maclean J.A. Charo I.F. Luster A.D. J. Exp. Med. 1997; 185: 99-109Crossref PubMed Scopus (224) Google Scholar). In addition, only chemokines of the MCP family (MCP-1, -2, -3, -4, and -5) appear to activate CCR2, although CCR2 agonists can also bind and activate other receptors, since MCP-3 activates CCR1 (16Franci C. Wong L.M. Van Damme J. Proost P. Charo I.F. J. Immunol. 1995; 154: 6511-6517PubMed Google Scholar, 17Combadiere C. Ahuja S.K. Van Damme J. Tiffany H.L. Gao J. Murphy P.M. J. Biol. Chem. 1995; 270: 29671-29675Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) and MCP-3 and MCP-4 activate CCR3 (18Garcia-Zepeda E.A. Combadiere C. Rothenberg M.E. Sarafi M.N. Lavigne F. Hamid Q. Murphy P.M. Luster A.D. J. Immunol. 1996; 157: 5613-5626PubMed Google Scholar, 19Stellato C.P. Collins P. Ponath P.D. Soler D. Newman W. La Rosa G. Li H. White J. Schwiebert L.M. Bickel C. Liu M. Bochner B.S. Williams T.J. Schleimer R.P. J. Clin. Invest. 1997; 99: 926-936Crossref PubMed Scopus (170) Google Scholar). Studies with CCR2−/− mice have recently revealed, however, that MCP-1 initiates cellular responses primarily through binding to CCR2 (21Boring L. Gosling J. Chensue S.W. Kunkel S.L. Farese R.V. Broxmeyer H.E. Charo I.F. J. Clin. Invest. 1997; 100: 2552-2561Crossref PubMed Scopus (888) Google Scholar). Analysis of the signal transduction pathways activated by MCP-1 has revealed pertussis toxin-sensitive phospholipase C activation (22Kuang Y. Wu Y. Jiang H. Wu D. J. Biol. Chem. 1996; 271: 3975-3978Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), elevation of intracellular calcium (14Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2752-2756Crossref PubMed Scopus (653) Google Scholar, 23Sozzani S. Molino M. Locati M. Luini W. Cerletti C. Vecchi A. Mantovani A. J. Immunol. 1993; 150: 1544-1553PubMed Google Scholar), and inhibition of adenyl cyclase (24Myers S.J. Wong L.M. Charo I.F. J. Biol. Chem. 1995; 270: 5786-5792Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Interestingly, both CCR2A and CCR2B can both couple to the Gi-Gβγ-PLCβ2 pathway, but these receptors demonstrate an interesting specificity in their coupling to the α-subunits of the Gq class. Hence, CCR2B couples to both Gα16 and Gα14, whereas CCR2A cannot couple to either Gα14 or Gα16 (22Kuang Y. Wu Y. Jiang H. Wu D. J. Biol. Chem. 1996; 271: 3975-3978Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Other signaling events downstream of MCP-1 remain relatively poorly defined. In this respect, it is interesting to note that the related CC chemokine RANTES has been demonstrated to stimulate the tyrosine phosphorylation of a number of proteins in a T-cell clone (25Bacon K.B. Szabo M.C. Yssel H. Bolen J.B. Schall T.J. J. Exp. Med. 1996; 184: 873-882Crossref PubMed Scopus (133) Google Scholar, 26Bacon K.B. Premack B.A. Gardner P. Schall T.J. Science. 1995; 269: 1727-1730Crossref PubMed Scopus (420) Google Scholar) and to activate the protein-tyrosine kinase (PTK)/src homology domain 2-coupled phosphatidylinositol (PI) 3-kinase (27Turner L. Ward S.G. Westwick J. J. Immunol. 1995; 155: 2437-2444PubMed Google Scholar), a member of the class 1A family of the phosphatidylinositol 3-kinases (28Vanhaesebroeck B. Leevers S.A. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (835) Google Scholar). The prototypical class 1A PI 3-kinase consists of an 85-kDa regulatory subunit responsible for protein-protein interactions via protein tyrosine phosphate-binding src homology domains and a catalytic 110-kDa subunit (28Vanhaesebroeck B. Leevers S.A. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (835) Google Scholar). PI 3-kinase is now regarded as an important intracellular signal that is upstream of a variety of responses including insulin-stimulated glucose uptake (29Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L.R. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. J. Biol. Chem. 1994; 91: 7415-7419Google Scholar), membrane ruffling (30Wennstrom S. Hawkins P.T. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L.R. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar), superoxide production (31Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1054) Google Scholar), activation of p70 S6 kinase (32Chung J. Grammer T. Lemon C. Kazlauskas A. Blenis J. Nature. 1994; 370: 71-73Crossref PubMed Scopus (656) Google Scholar), and activation of Akt/protein kinase B (33Burgering B.T. Coffer P.J. Nature. 1995; 376: 599-601Crossref PubMed Scopus (1884) Google Scholar). A G protein-coupled PI 3-kinase, namely PI 3-kinase γ has also been identified (34Stephens L.R. Eguinoa A. Erdjument-Bromage 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 (494) Google Scholar, 35Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (641) Google Scholar). PI 3-kinase γ is, to date, the only characterized member of the class 1B G protein-coupled PI 3-kinase family and consists of a unique 101-kDa regulatory subunit and a distinct 110-kDa catalytic subunit termed p110γ (28Vanhaesebroeck B. Leevers S.A. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (835) Google Scholar, 33Burgering B.T. Coffer P.J. Nature. 1995; 376: 599-601Crossref PubMed Scopus (1884) Google Scholar, 34Stephens L.R. Eguinoa A. Erdjument-Bromage 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 (494) Google Scholar). Nevertheless, there is some evidence that G protein-coupled receptors, such as fMLP receptors, are able to activate the p85/p110 PI 3-kinase (36Stephens L. Eguinoa A. Corey S. Jackson T.R. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (137) Google Scholar, 37Kurosu H. Maehama T. Okada T. Yamamoto T. Hoshino S. Fukui Y. Ui M. Hazeki O. Katada T. J. Biol. Chem. 1996; 272: 24252-24256Abstract Full Text Full Text PDF Scopus (235) Google Scholar). In this respect, the p85/p110 heterodimer has been demonstrated to be synergistically activated by the βγ subunits of G proteins and by phosphotyrosyl peptide (37Kurosu H. Maehama T. Okada T. Yamamoto T. Hoshino S. Fukui Y. Ui M. Hazeki O. Katada T. J. Biol. Chem. 1996; 272: 24252-24256Abstract Full Text Full Text PDF Scopus (235) Google Scholar). The class I PI 3-kinases can potentially generate three lipid products, namely phosphatidylinositol 3-monophosphate (PI 3-P), phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2), and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3), which are collectively known as D-3 phosphatidylinositol lipids (reviewed in Refs. 38Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar and 39Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1229) Google Scholar). To date, PI 3,4-P2 and PI 3,4,5-P3 are regarded as signaling molecules, whereas PI 3-P is thought to regulate membrane trafficking (38Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar, 39Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1229) Google Scholar). The PI 3-kinase family is completed by the class II C2 domain-containing PI 3-kinases and the class III PtdIns-specific 3-kinases (28Vanhaesebroeck B. Leevers S.A. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (835) Google Scholar). The 190-kDa PI3K-C2α is a novel human member of the class II PI 3-kinase family that, in contrast to members of the other PI 3-kinase classes, is refractory to inhibition by the PI 3-kinase inhibitors wortmannin and LY294002 (40Domin J. Pages F. Volinia S. Rittenhouse S.E. Zvelebil M.J. Stein R.C. Waterfield M.D. Biochem. J. 1997; 326: 139-147Crossref PubMed Scopus (219) Google Scholar). In addition, PI3K-C2α utilizes predominantly PI and phosphatidylinositol 4-monophosphate as substrates in vitro, but when presented with phosphatidylserine it can also phosphorylate PI 4,5-bisphosphate (40Domin J. Pages F. Volinia S. Rittenhouse S.E. Zvelebil M.J. Stein R.C. Waterfield M.D. Biochem. J. 1997; 326: 139-147Crossref PubMed Scopus (219) Google Scholar). Given the functional role of MCP-1 on superoxide generation and chemotaxis (4Carr M.W. Roth S.J. Luther E. Rose S.S. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3652-3656Crossref PubMed Scopus (1049) Google Scholar, 5Zachariae C.O.C. Anderson A.O. Thompson H.L. Appella E. Mantovani A. Oppenheim J.J. Matsushima K. J. Exp. Med. 1990; 171: 2177-2182Crossref PubMed Scopus (202) Google Scholar), it is interesting to note that the PI 3-kinase inhibitor wortmannin inhibits fMLP-stimulated superoxide release (31Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1054) Google Scholar), interleukin-8-stimulated neutrophil chemotaxis (41Knall C. Worthen G.S. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3052-3057Crossref PubMed Scopus (276) Google Scholar), and RANTES-stimulated chemotaxis of T-lymphocyte (27Turner L. Ward S.G. Westwick J. J. Immunol. 1995; 155: 2437-2444PubMed Google Scholar). In this study, therefore, we have investigated the possible involvement of PI 3-kinase(s) in MCP-1 signal transduction and chemotaxis using the THP-1 monocytic cell line. Human recombinant MCP-1 was purchased from Peprotech (Rocky Hill, NJ). Mouse p85α monoclonal antibody (mAb) was a generous gift from Doreen Cantrell (Imperial Cancer Research Fund, London). 4G10 anti-phosphotyrosine mAb was purchased from Upstate Biotechnology. All cell culture reagents and pertussis toxin were purchased from Life Technologies, Inc. Wortmannin and standard phosphatidylinositol lipids were purchased from Sigma as was the PY20 anti-phosphotyrosine antibody. [γ-32P]ATP (3000 Ci/mmol) and [32P]orthophosphate (8500–9120 Ci/mmol) were from NEN Life Science Products. All other reagents were purchased from Sigma. An N-terminal fragment was expressed as a glutathioneS-transferase fusion protein in the following manner. Nucleotides 4–1011 of the PI3K-C2α cDNA sequence (encoding residues 2–337) were amplified by PCR using complementary oligonucleotides, which would incorporate a SmaI and anEcoRI restriction site at its 5′- and 3′-end, respectively. Following digestion with SmaI/EcoRI, this cDNA was ligated into a pGex2T expression vector (Amersham Pharmacia Biotech) to allow its in frame expression at the C terminus of glutathioneS-transferase. This plasmid was used to transformEpicurian coli XL-1 cells, and production of the glutathioneS-transferase fusion was induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside (30 °C for 4 h). Cells were harvested and lysed in PBS containing 1% Triton X-100, 2 mm EDTA, 5 mm benzamidine, 1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, 50 milli-trypsin inhibitory units/ml aprotonin, 50 μm pepstatin, and 50 μm leupeptin (PBST buffer) at 4 °C. The fusion protein was affinity-purified using glutathione-Sepharose beads (4 h, 40 °C), and after washing, it was eluted from the beads upon the addition of 10 mmglutathione, 150 mm NaCl in 100 μm Tris, pH 8.0. Glutathione was removed by dialysis against 50 mmTris, pH 7.4, 150 mm NaCl, and 1 mmdithiothreitol at 4 °C. Aliquots of the expressed fusion protein were frozen and injected into rabbits (100 μg) at monthly intervals. One week after each injection, serum was tested for its ability to immunoprecipitate and Western blot recombinant PI3K-C2α protein and PI3K-C2α immunoreactivity from cell lysates. The resulting antisera displayed no cross-reaction upon Western blotting against a range of other recombinant PI 3-kinase protein standards. 2J. Domin and M. Waterfield, unpublished observations. The human monocytic cell line THP-1 expressing CCR2 was obtained from the European Collection of Animal Cell Cultures (14Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2752-2756Crossref PubMed Scopus (653) Google Scholar). The cells were cultured in humidified incubators at 37 °C, 5% (v/v) CO2 in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. 1 × 108 cells were labeled with 1 mCi of [32P]orthophosphate (8500–9120 Ci/mmol; NEN Life Science Products) as described (42Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16633Abstract Full Text PDF PubMed Google Scholar). 32P-Labeled THP-1 cells were aliquoted at 107/120 μl and stimulated as described in the figure legends, and the phospholipids were extracted with 700 μl of chloroform:methanol:H2O (32.6, 65.3, and 2.1% (v/v/v), respectively) (42Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16633Abstract Full Text PDF PubMed Google Scholar). The samples were deacylated and analyzed by anion exchange HPLC analysis using a Partisphere SAX column (Whatman) (42Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16633Abstract Full Text PDF PubMed Google Scholar). The eluate was fed into a Canberra Packard A-500 Flo-One on-line radiodetector, and the results were analyzed by the Flo-One data program (Radiomatic). Eluted peaks were compared with retention times for standards prepared from 3H-labeled phosphatidylinositol lipids (Amersham Pharmacia Biotech) and32P-labeled D-3 phosphatidylinositols described elsewhere (42Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16633Abstract Full Text PDF PubMed Google Scholar). 2 × 107 cells/ml were equilibrated for 10 min at 37 °C and then stimulated in RPMI 1640 medium as described in the figure legends. Reactions were terminated by pelleting cells in a microcentrifuge for 10 s and aspirating supernatant, followed by the addition of 0.5 ml of ice-cold lysis buffer (1% (v/v) Nonidet P-40, 100 mmNaCl, 20 mm Tris (pH 7.4), 10 mm iodoacetamide, 10 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 10 μg/ml β-glycerophosphate, and 1 mm sodium orthovanadate). Lysates were rotated at 4 °C for 15 min, followed by centrifugation at 14,000 rpm. The supernatants were precleared, and immunoprecipitation was performed as described (43Ward S.G. Reif K. Ley S. Fry M.J. Waterfield M.D. Cantrell D.A. J. Biol. Chem. 1992; 267: 23862-23869Abstract Full Text PDF PubMed Google Scholar, 44Wright K. Ward S.G. Kolios G. Westwick J. J. Biol. Chem. 1997; 272: 12626-12633Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) using either p85α mAb (1 μg/ml) precoupled to protein G-Sepharose beads (Pharmacia Biotech Inc.) or PI3K-C2α polyclonal antibody precoupled to protein A-Sepharose beads. Immunoprecipitates were washed and subjected to in vitro lipid kinase assays as described (43Ward S.G. Reif K. Ley S. Fry M.J. Waterfield M.D. Cantrell D.A. J. Biol. Chem. 1992; 267: 23862-23869Abstract Full Text PDF PubMed Google Scholar,44Wright K. Ward S.G. Kolios G. Westwick J. J. Biol. Chem. 1997; 272: 12626-12633Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) using a lipid mixture of 100 μl of 0.1 mg/ml PI and 0.1 mg/ml phosphatidylserine dispersed by sonication in 20 mm HEPES, pH 7.0, and 1 mm EDTA. The reaction was initiated by the addition of 10 μCi of [γ-32P]ATP (3000 Ci/mmol; NEN Life Science Products) and 100 μm ATP to the immunoprecipitates suspended in 80 μl of kinase buffer (5 mm MgCl2, 0.25 mm EDTA, 20 mm HEPES, pH 7.4). The reaction was terminated after 15 min, and the resulting phospholipids were then separated by TLC (43Ward S.G. Reif K. Ley S. Fry M.J. Waterfield M.D. Cantrell D.A. J. Biol. Chem. 1992; 267: 23862-23869Abstract Full Text PDF PubMed Google Scholar,44Wright K. Ward S.G. Kolios G. Westwick J. J. Biol. Chem. 1997; 272: 12626-12633Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The TLC plates were stained with iodine to confirm even extraction of substrate lipid between individual samples, and32P-labeled PI 3-P was visualized by autoradiography (43Ward S.G. Reif K. Ley S. Fry M.J. Waterfield M.D. Cantrell D.A. J. Biol. Chem. 1992; 267: 23862-23869Abstract Full Text PDF PubMed Google Scholar,44Wright K. Ward S.G. Kolios G. Westwick J. J. Biol. Chem. 1997; 272: 12626-12633Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). 2 × 107 cells/ml resuspended in RPMI 1640 were equilibrated at 37 °C for 10 min and stimulated as described in the figure legends, and cell lysates were prepared (43Ward S.G. Reif K. Ley S. Fry M.J. Waterfield M.D. Cantrell D.A. J. Biol. Chem. 1992; 267: 23862-23869Abstract Full Text PDF PubMed Google Scholar, 44Wright K. Ward S.G. Kolios G. Westwick J. J. Biol. Chem. 1997; 272: 12626-12633Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Aliquots of cell lysate (20 μl) or immunoprecipitates generated using either anti-phosphotyrosine mAb PY20 (1 μg/ml) or anti-p85 (1 μg/ml) antibodies were boiled in Laemmli buffer and electrophoresed through 7.5% (v/v) acrylamide gels by SDS-PAGE, and the proteins were transferred by electroblotting onto nitrocellulose (Schleicher & Schuell) as described previously (44Wright K. Ward S.G. Kolios G. Westwick J. J. Biol. Chem. 1997; 272: 12626-12633Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The blots were probed with the anti-PI3K-C2α polyclonal antibody (1:500 dilution) or anti-phosphotyrosine mAb 4G10 (0.1 μg/ml) as indicated in the appropriate figure legends, and the proteins were visualized by a chemiluminescence detection system (ECL; Amersham Pharmacia Biotech) with goat anti-rabbit or goat anti-mouse Ig (0.1 μg/ml) horseradish peroxidase conjugate, respectively, as a secondary antibody (Dako). THP-1 cell chemotaxis was examined using a 96-well chemotaxis chamber (Neuro Probe, Cabin John, MD). The wells of the 96-well plate were filled with 380 μl of chemoattractant diluted in RPMI 1640 containing 0.1% bovine serum albumin and covered with an adhesive polyvinylpyrrolidine-free polycarbonate membrane (5-μm pore size). 5 × 105 cells were added to each upper well in a volume of 200 μl, and the chamber was incubated at 37 °C for 2 h. The cell suspension was subsequently aspirated off, and 200 μl of Versene (Life Technologies Inc., Paisley, UK) was added to each well. After a 20-min incubation at 4 °C, the 96-well plate and membrane were centrifuged at 1200 rpm for 10 min, the supernatant was removed, and the cells were resuspended in 100 μl of RPMI containing 0.1% bovine serum albumin. THP-1 cell migration was assessed by adding 20 μl of Cell Titer 96 AQueoussolution (Promega, UK) to each well. After a 2-h incubation at 37 °C, the plate was read at a wavelength of 490 nm, subtracting the readings at a reference wavelength of 650 nm to reduce the background contributed by nonspecific absorbance. MCP-1 induces a rapid and transient activation of phospholipase C in THP-1 cells (23Sozzani S. Molino M. Locati M. Luini W. Cerletti C. Vecchi A. Mantovani A. J. Immunol. 1993; 150: 1544-1553PubMed Google Scholar, 45Turner S.J. Westwick J. Br. J. Pharmacol. 1995; 114: 211PGoogle Scholar). In this study, we have used 32P-labeled THP-1 cells to investigate the effect of MCP-1 stimulation on the activation of another signaling pathway, namely PI 3-kinase. Accordingly, treatment of THP-1 cells with MCP-1 resulted in the concentration-dependent formation of PI 3,4,5-P3 (Fig. 1 A). The MCP-1-induced increase in PI 3,4,5-P3 exhibited bell-shaped characteristics with the maximum response observed in the presence of 60 nm MCP-1 (Fig. 1 A). Furthermore, the MCP-1-stimulated formation of PI 3,4,5-P3 was extremely rapid and transient, since it was detectable 5 s after stimulation and had returned to basal levels 2 min after MC
Referência(s)