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• W2001044562 abstract "The α-chemokine stromal cell-derived factor (SDF)-1α binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation. The signaling pathways that mediate the effects of SDF-1α are not well characterized. We studied events following SDF-1α binding to CXCR-4 in a model murine pre-B cell line transfected with human CXCR-4. There was enhanced tyrosine phosphorylation and association of components of focal adhesion complexes such as the related adhesion focal tyrosine kinase, paxillin, and Crk. We also observed activation of phosphatidylinositol 3-kinase. Wortmannin, a selective inhibitor of phosphatidylinositol 3-kinase, partially inhibited the SDF-1α-induced migration and tyrosine phosphorylation of paxillin. SDF-1α treatment selectively activated p44/42 mitogen-activated protein kinase (Erk 1 and Erk 2) and its upstream kinase mitogen-activated protein kinase kinase but not p38 mitogen-activated protein kinase, c-Jun amino-terminal kinase or mitogen activated protein kinase kinase. We also observed that SDF-1α treatment increased NF-κB activity in nuclear extracts from the CXCR-4 transfectants. Taken together, these studies revealed that SDF-1α activates distinct signaling pathways that may mediate cell growth, migration, and transcriptional activation. The α-chemokine stromal cell-derived factor (SDF)-1α binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation. The signaling pathways that mediate the effects of SDF-1α are not well characterized. We studied events following SDF-1α binding to CXCR-4 in a model murine pre-B cell line transfected with human CXCR-4. There was enhanced tyrosine phosphorylation and association of components of focal adhesion complexes such as the related adhesion focal tyrosine kinase, paxillin, and Crk. We also observed activation of phosphatidylinositol 3-kinase. Wortmannin, a selective inhibitor of phosphatidylinositol 3-kinase, partially inhibited the SDF-1α-induced migration and tyrosine phosphorylation of paxillin. SDF-1α treatment selectively activated p44/42 mitogen-activated protein kinase (Erk 1 and Erk 2) and its upstream kinase mitogen-activated protein kinase kinase but not p38 mitogen-activated protein kinase, c-Jun amino-terminal kinase or mitogen activated protein kinase kinase. We also observed that SDF-1α treatment increased NF-κB activity in nuclear extracts from the CXCR-4 transfectants. Taken together, these studies revealed that SDF-1α activates distinct signaling pathways that may mediate cell growth, migration, and transcriptional activation. human immunodeficiency virus stromal cell-derived factor phosphate-buffered saline related adhesion focal tyrosine kinase fluorescence-activated cell sorter fetal calf serum total cell lysates room temperature polyacrylamide gel electrophoresis mitogen-activated protein MAP kinase phosphatidylinositol 3-kinase c-Jun amino-terminal kinase: MKK, mitogen-activated protein kinase kinase. Chemokines and their receptors have recently received considerable attention because of their emerging role in immune and inflammatory responses, hematopoiesis, and HIV1 infection (1Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, 2Premack B.A. Schall T.J. Nat. Med. 1996; 2: 1174-1178Crossref PubMed Scopus (572) Google Scholar, 3Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar, 4Gerard C. Gerard N.P. Curr. 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The CXC chemokine, stromal cell-derived factor (SDF-1α), was first cloned from mouse bone marrow and characterized as a pre-B cell growth-stimulating factor (8Tashiro K. Tada H. Heilker R. Shirozu M. Nakano T. Honjo T. Science. 1993; 261: 600-603Crossref PubMed Scopus (638) Google Scholar, 9Shirozu M. Nakano T. Inazawa J. Tashiro K. Tada H. Shinohara T. Honjo T. Genomics. 1995; 28: 495-500Crossref PubMed Scopus (537) Google Scholar, 10Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1289) Google Scholar, 11Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (2020) Google Scholar, 12Aiuti A. Webb I.J. Bleul C. Springer T. Gutierrez-Ramos J.C. J. Exp. Med. 1997; 185: 111-120Crossref PubMed Scopus (1206) Google Scholar, 13Sanchez X. Cousins-Hodges B. Aguilar T. Gosselink P. Lu Z. Navarro J. J. Biol. Chem. 1997; 272: 27529-27531Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Two isoforms, SDF-1α and SDF-1β, have been identified that are encoded by a single gene and arise by alternative splicing (9Shirozu M. Nakano T. Inazawa J. Tashiro K. Tada H. Shinohara T. Honjo T. Genomics. 1995; 28: 495-500Crossref PubMed Scopus (537) Google Scholar). SDF-1α is widely expressed and, in addition to its effects on pre-B cells, is a potent chemotactic factor for monocytes, T-lymphocytes, and CD34+ human progenitor cells (8Tashiro K. Tada H. Heilker R. Shirozu M. Nakano T. Honjo T. Science. 1993; 261: 600-603Crossref PubMed Scopus (638) Google Scholar, 9Shirozu M. Nakano T. Inazawa J. Tashiro K. Tada H. Shinohara T. Honjo T. Genomics. 1995; 28: 495-500Crossref PubMed Scopus (537) Google Scholar, 10Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1289) Google Scholar, 11Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (2020) Google Scholar, 12Aiuti A. Webb I.J. Bleul C. Springer T. Gutierrez-Ramos J.C. J. Exp. Med. 1997; 185: 111-120Crossref PubMed Scopus (1206) Google Scholar, 13Sanchez X. Cousins-Hodges B. Aguilar T. Gosselink P. Lu Z. Navarro J. J. Biol. Chem. 1997; 272: 27529-27531Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Knock-out mice lacking SDF-1α protein show abnormalities in B cell lymphopoiesis, bone marrow myelopoiesis, and also have nonfatal ventricular septal defects (11Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (2020) Google Scholar). SDF-1α was recently shown to be a ligand for the chemokine receptor CXCR-4 (14Loetscher M. Geiser T. O'Reilly T. Zwahlen R. Baggiolini M. Moser B. J. Biol. Chem. 1994; 269: 232-237Abstract Full Text PDF PubMed Google Scholar, 15Bleul C.C. Farzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1755) Google Scholar, 16Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J.L. Arenzana-Seisdedos F. Schwartz O. Heard J.M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1484) Google Scholar, 17Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3643) Google Scholar, 18Heesen M. Berman M.A. Benson J.D. Gerard C. Dorf M.E. J. Immunol. 1996; 157: 5455-5460PubMed Google Scholar). CXCR-4 is a seven transmembrane G-protein-coupled receptor (19Haribabu B. Richardson R.M. Fisher I. Sozzani S. Peiper S.C. Horuk R. Ali H. Snyderman R. J. Biol. Chem. 1997; 272: 28726-28731Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 20Davis C.B. Dikic I. Unutmaz D. Hill C.M. Arthos J. Siani M.A. Thompson D.A. Schlessinger J. Littman D.R. J. Exp. Med. 1997; 186: 1793-1798Crossref PubMed Scopus (344) Google Scholar). Recently, it has been shown that CXCR-4 expression can be regulated by receptor phosphorylation-dependent and -independent mechanisms (19Haribabu B. Richardson R.M. Fisher I. Sozzani S. Peiper S.C. Horuk R. Ali H. Snyderman R. J. Biol. Chem. 1997; 272: 28726-28731Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). A diversity of white cells including peripheral blood lymphocytes, monocytes, thymocytes, pre-B cells, and dendritic and endothelial cells express the CXCR-4 receptor (14Loetscher M. Geiser T. O'Reilly T. Zwahlen R. Baggiolini M. Moser B. J. Biol. Chem. 1994; 269: 232-237Abstract Full Text PDF PubMed Google Scholar,21Bleul C.C. Wul L. Hoxie J.A. Springer T.A. Mackay C.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1925-1930Crossref PubMed Scopus (952) Google Scholar, 22Sallusto F. Lenig D. Mackay C.R. Lanzavecchia A. J. Exp. Med. 1998; 187: 875-883Crossref PubMed Scopus (1378) Google Scholar, 23Forster R. Kremmer E. Schubel A. Breitfeld D. Kleinschmidt A. Nerl C. Bernhardt G. Lipp M. J. Immunol. 1998; 160: 1522-1531PubMed Google Scholar, 24Hesselgesser J. Liang M. Hoxie J. Greenberg M. Brass L.F. Orsini M.J. Taub D. Horuk R. J. Immunol. 1998; 160: 877-883PubMed Google Scholar, 25Gupta S.K. Lysko P.G. Pillarisetti K. Ohlstein E. Stadel J.M. J. Biol. Chem. 1998; 273: 4282-4287Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 26Yi Y. Rana S. Turner J.D. Gaddis N. Collman R.G. J. Virol. 1998; 72: 772-777Crossref PubMed Google Scholar). CXCR-4 has been shown to act as a co-receptor for the binding of T cell tropic HIV-1 strains (6Moore J.P. Trkola A. Dragic T. Curr. Opin. Immunol. 1997; 9: 551-562Crossref PubMed Scopus (455) Google Scholar, 15Bleul C.C. Farzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1755) Google Scholar, 16Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J.L. Arenzana-Seisdedos F. Schwartz O. Heard J.M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1484) Google Scholar, 17Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3643) Google Scholar). SDF-1α and its various analogues can inhibit CXCR-4-mediated HIV-1 infection in vitro (27Ueda H. Siani M.A. Gong W. Thompson D.A. Brown G.G. Wang J.M. J. Biol. Chem. 1997; 272: 24966-24970Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 28Murakami T. Nakajima T. Koyanagi Y. Tachibana K. Fujii N. Tamamura H. Yoshida N. Waki M. Matsumoto A. Yoshie O. Kishimoto T. Yamamoto N. Nagasawa T. J. Exp. Med. 1997; 186: 1389-1393Crossref PubMed Scopus (365) Google Scholar). Despite the increasingly prominent role of SDF-1α and its receptor CXCR-4 in the regulation of cell proliferation, migration, and HIV infection, relatively little is known about the signaling pathways that may mediate these effects (19Haribabu B. Richardson R.M. Fisher I. Sozzani S. Peiper S.C. Horuk R. Ali H. Snyderman R. J. Biol. Chem. 1997; 272: 28726-28731Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 20Davis C.B. Dikic I. Unutmaz D. Hill C.M. Arthos J. Siani M.A. Thompson D.A. Schlessinger J. Littman D.R. J. Exp. Med. 1997; 186: 1793-1798Crossref PubMed Scopus (344) Google Scholar). In this study, we show that SDF-1α stimulation in CXCR-4 transfectants results in the increased phosphorylation of focal adhesion components, including the related adhesion focal tyrosine kinase (RAFTK/Pyk2), Crk, and paxillin. SDF-1α treatment activated the p44/42 MAP kinases (Erk 1 and 2), PI-3 kinase, and NF-κB. These studies indicate that activation of CXCR-4 results in modulation of signaling molecules and transcription factors that mediate changes in the cytoskeletal apparatus and also regulate cell growth. RAFTK antibodies were generated using C domain glutathione S-transferase fusion proteins as described previously (29Li J. Avraham H. Rogers R.A. Raja S. Avraham S. Blood. 1996; 88: 417-428Crossref PubMed Google Scholar). Serum R-4250 was chosen for further studies based on its titer in enzyme-linked immunosorbent assay. This antiserum does not cross-react with FAK and recognizes both human and murine forms of RAFTK. Monoclonal anti-phosphotyrosine antibody (4G10) was a generous gift from Dr. Brian Druker (Oregon Health Sciences University, Portland, OR). Purified antibodies to JNK, p38 MAP kinase, p44/42 MAPK, and recombinant GST-c-Jun amino-terminal proteins (1–79 amino acids) were obtained from Santa Cruz Laboratories (Santa Cruz, CA). Antibodies to paxillin and Crk were obtained from Transduction Laboratories, Inc. (Lexington, KY). Monoclonal antibodies to CXCR-4 and the isotype control were from PharMingen (San Diego, CA). Electrophoresis reagents were obtained from Bio-Rad. The protease inhibitors leupeptin and α1-antitrypsin as well as all other reagents were obtained from Sigma. Wortmannin was obtained from Calbiochem, and the nitrocellulose membrane was from Bio-Rad. Indo-1 acetoxymethyl ester (Indo-1 AM) was purchased from Molecular Probes (Eugene, OR). We used a murine pre-B lymphoma cell line, L1.2, for the transfection studies. CXCR-4 cDNA, tagged at the amino terminus with a Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), was subcloned into the pcDNAIII expression vector. The DNA was stably transfected into the L1.2 cells as described (30Ponath P.D. Qin S. Post T.W. Wang J. Wu L. Gerard N.P. Newman W. Gerard C. Mackay C.R. J. Exp. Med. 1996; 183: 2437-2448Crossref PubMed Scopus (553) Google Scholar, 31Wu L. Ruffing N. Shi X. Newman W. Soler D. Mackay C.R. Qin S. J. Biol. Chem. 1996; 271: 31202-31209Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 32Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffing N. Borsetti A. Cardoso A.A. Desjardin E. Newman W. Gerard C. Sodorski J. Nature. 1996; 384: 179-181Crossref PubMed Scopus (1083) Google Scholar), and G418-selective medium was used to select for transfectants. Cell-surface expression of CXCR-4 on the transfectants was confirmed by FACS analysis. The L1.2 cells were grown at 37 °C in 5% CO2 in RPMI 1640 with 10% fetal calf serum (FCS), 2 mm glutamine, 1 mm sodium pyruvate, 50 μg/ml penicillin, 50 μg/ml streptomycin, and 55 μmβ-mercaptoethanol. The CXCR-4 transfectants were grown in this RPMI 1640 media containing 0.8 mg/ml Geneticin (G418) (Life Technologies, Inc.). The CXCR-4 L1.2 transfectants (1 × 106) were washed twice with phosphate-buffered saline (PBS), resuspended in 100 μl of PBS containing 5% FCS and 5 μg/ml PE-labeled CXCR-4 antibody or isotype control (PharMingen), and then incubated for 30 min at 4 °C. The cells were washed twice with ice-cold PBS, 5% FCS, resuspended in 500 μl of PBS, 5% FCS buffer, and then analyzed by flow cytometry to determine the levels of surface expression of these receptors. The CXCR-4 transfectants were washed with RPMI 1640 and resuspended at 10 × 106 cells/ml in the RPMI medium. The cells were loaded with Indo-1 AM (Molecular Probes) by adding 5 μl of working Indo-1 solution to the 10 × 106 cells that were suspended in 1 ml of RPMI solution and incubated for 45 min at 37 °C. Cells were diluted to 1 × 106/ml, treated with SDF-1α, and analyzed for calcium mobilization by flow cytometry (Coulter Electronics, Hialeah, FL) as described (33Ganju R.K. Dutt P. Wu L. Newman W. Avraham H. Avraham S. Groopman J.E. Blood. 1998; 91: 791-797Crossref PubMed Google Scholar). Calcium flux assays and all other subsequent signaling assays were repeated at least three times. Cells were washed twice with RPMI 1640 (Life Technologies, Inc.) and resuspended at 10 × 106cells/ml in the same medium. Cells were starved for 4 h at 37 °C and then stimulated with different concentrations of SDF-1α at 37 °C for various periods. After stimulation, cells were lysed in modified RIPA buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, leupeptin, and pepstatin, 10 mm sodium vanadate, 10 mm sodium fluoride, and 10 mm sodium pyrophosphate). Total cell lysates (TCL) were clarified by centrifugation at 10,000 × g for 10 min. Protein concentrations were determined by protein assay (Bio-Rad). Cell lysis, immunoprecipitation, immunoblotting, kinase assays, and autophosphorylation assays were carried out as described below. For the immunoprecipitation studies, identical amounts of protein from each sample were clarified by incubation with protein A-Sepharose CL-4B or Gammabind plus Sepharose (Amersham Pharmacia Biotech) for 1 h at 4 °C. The beads were removed by brief centrifugation, and the solution was incubated with different primary antibodies for each experiment for 4 h or overnight at 4 °C. Antibody-antigen complexes were immunoprecipitated by incubation with 50 μl of protein A-Sepharose or Gammabind Sepharose (10% suspension) for 4 h at 4 °C. The Sepharose beads were washed three times with modified RIPA buffer and one time with PBS to remove the nonspecifically bound proteins. Bound proteins were solubilized in 40 μl of 2× Laemmli buffer and further analyzed by immunoblotting. The samples were separated on SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were blocked and probed with primary antibody for 3 h at room temperature (RT) or 4 °C overnight. Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescent (ECL) system (Amersham Pharmacia Biotech). Monoclonal antibody (4G10, IgG2a) was used for Western blot analysis of the phosphotyrosine protein. In vitro kinase assays were performed as described earlier (34Ganju R.K. Hatch W.C. Avraham H. Ona M.A. Druker B. Avraham S. Groopman J.E. J. Exp. Med. 1997; 185: 1055-1063Crossref PubMed Scopus (94) Google Scholar). The cell lysates immunoprecipitated with RAFTK antiserum were washed twice with RIPA buffer and once in kinase buffer (20 mm HEPES, pH 7.4, 50 mm NaCl, 5 mm MgCl2, 5 mm MnCl2, 100 mmNa3VO4). For the in vitro kinase assays, the immune complex was incubated in kinase buffer containing 25 μg of poly(Glu:Tyr) (4:1), 20–50 kDa (Sigma), and 5 μCi of [γ-32P]ATP at RT for 30 min. The reaction was stopped by adding 2× SDS sample buffer and boiling the sample for 5 min at 100 °C. Proteins were then separated on 10% SDS-PAGE and detected by autoradiography. Normal rabbit serum was used as a negative control. The autophosphorylation assay was carried out by incubating the immune complex in kinase buffer containing 5 μCi of [γ-32P]ATP at RT for 30 min. The reaction was stopped by adding 4× SDS sample buffer and by boiling the sample for 5 min. Proteins were then separated on SDS-PAGE and detected by autoradiography. The JNK assay was performed as described earlier (35Liu Z.Y. Ganju R.K. Wang J.F. Schweitzer K. Weksler B. Avraham S. Groopman J.E. Blood. 1997; 90: 2253-2257Crossref PubMed Google Scholar). Briefly, cell lysates were immunoprecipitated with JNK antibody (Santa Cruz Biotechnology). The immune complexes were washed twice with RIPA buffer and once in kinase buffer (50 mm HEPES, pH 7.4, 10 mmMgCl2, 20 μm ATP). The complex was then incubated in kinase buffer containing recombinant GST-c-Jun 0.2 μg/μl (1–79 amino acids) (Santa Cruz Biotechnology) and 5 μCi of [γ-32P]ATP for 10 min at RT. The reaction was terminated by adding 2× SDS sample buffer and boiling the sample for 5 min at 100 °C. Proteins were separated on 12% SDS-PAGE and detected by autoradiography. Rabbit IgG was used as a negative control. For the p44/42 and p38 MAP kinase assays, cell lysates from unstimulated or stimulated cells were immunoprecipitated with Erk 1 and Erk 2 (p44/42) or p38 MAP kinase antibody (Santa Cruz Biotechnology). The immune complexes were washed twice with RIPA buffer and once in kinase buffer (50 mm HEPES, pH 7.4, 10 mm MgCl2and 20 μm ATP). The complex was then incubated in kinase buffer containing 7 μg of myelin basic protein (Upstate Biotechnology, Lake Placid, NY) and 5 μCi of [γ-32P]ATP for 20 min at 30 °C. Proteins were separated on 15% SDS-PAGE and detected by autoradiography. Rabbit IgG was used as a negative control. For MKK-1 or MKK-4 kinase assays, the cell lysates were immunoprecipitated with MKK-1 or MKK-4 antibodies (Santa Cruz Biotechnology). The immunoprecipitates were washed with RIPA buffer and kinase buffer (50 mm HEPES, pH 7.4, 10 mm MgCl2, 20 μm ATP). The immune complexes were incubated in kinase buffer containing 1.0 μg of recombinant p42 MAP kinase fusion protein (Santa Cruz) for the MKK-1 assay or 1.0 μg of JNK fusion protein-(1–384) (Santa Cruz) for the MKK-4 assay, and 5 μCi of [γ-32P]ATP for 20 min at 30 °C. Proteins were separated on 12% SDS-PAGE and detected by autoradiography. Rabbit IgG was used as a negative control. PI-3 kinase assays were performed as described (36Derman M.P. Chen J.Y. Spokes K.C. Songyang Z. Cantley L.G. J. Biol. Chem. 1996; 271: 4251-4255Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, equal amounts of protein from each sample were immunoprecipitated with either anti-phosphotyrosine antibody (4G10) or class-matched mouse IgG. Nonspecific binding was removed by washing the samples three times each with PBS containing 1% Nonidet P-40, followed by 0.5 mm Tris containing 0.5 mm lithium chloride and then by TE buffer. Samples were resuspended in 20 μl of TE buffer, 20 μl of phosphoinositol (10 μg, Avanti Polar Lipids, Alabaster, AL), and 10 μl of ATP mix (1 mm HEPES, 10 μm ATP, 1 μmMgCl2, and 5 μCi of [γ-32P]ATP) and incubated at RT for 10 min. The reaction was stopped by adding 60 μl of 2 mm HCl and 160 μl of chloroform:methanol (1:1 v/v). Lipids were separated on oxalate-impregnated silica TLC plates using a solvent system of chloroform:methanol:water:ammonium hydroxide (20%) (35:35:3.5:7), followed by autoradiography at −80 °C. The chemotaxis assay was performed in 24-well plates containing 5-μm porosity inserts (Costar Corp., Kennebunk, ME). Cells grown in RPMI 1640 medium containing 10% FCS were washed twice and suspended as 10 × 106 cells per ml in RPMI 1640 and H199 medium (1:1) containing 0.5% bovine serum albumin. Chemokines were then added to the bottom wells, and 100 μl (1 × 106) of cells were loaded onto the inserts. Cells migrating to the bottom well were collected after 2–4 h and counted on a flow cytometer. To assess the effect of wortmannin on migration, the cells were resuspended in medium containing different concentrations of wortmannin, and the chemotaxis assays were done as described above. Double-stranded oligonucleotides containing the consensus binding site for NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) were labeled with [γ-32P]ATP (3,000 Ci/mmol, NEN Life Science Products) using polynucleotide kinase (Promega, Madison, WI) according to established procedures. 10 μg of nuclear extract were incubated with labeled DNA (0.4 ng, 4,400 cpm) for 10 min at RT in the presence of DNA-binding buffer and 250 ng of poly(dI-dC) oligomer (Boehringer Mannheim) as described previously (37Yang Y.M. Rutberg S.E. Luo F.C. Spratt T.E. Halaban R. Ferrone S. Ronai Z. Cell Growth Differ. 1993; 4: 595-602PubMed Google Scholar). The complexes were then separated on a 7.5% polyacrylamide gel and autoradiographed. The results shown are representative of findings from three independent experiments. Human CXCR-4 cDNA was stably transfected into the murine pre-B lymphoma cell line, L1.2. Untransfected and transfected cell lines were analyzed for CXCR-4 expression. As shown in Fig. 1, CXCR-4 transfectants expressed high levels of the receptor in these transfected cells. Signal transduction by the binding of ligands to their cognate chemokine receptors involves characteristic calcium fluxes. To confirm that the CXCR-4 L1.2 cells expressing functional human CXCR-4 receptors retained this fundamental signaling property, the cells were treated with SDF-1α, and calcium fluxes were monitored by FACS analysis. SDF-1α treatment induced characteristic calcium fluxes in the CXCR-4 L1.2 cells (data not shown). RAFTK, a recently identified member of the focal adhesion kinase family, has been shown to be activated by various growth factors and chemokines (34Ganju R.K. Hatch W.C. Avraham H. Ona M.A. Druker B. Avraham S. Groopman J.E. J. Exp. Med. 1997; 185: 1055-1063Crossref PubMed Scopus (94) Google Scholar, 38Avraham S. London R. Fu Y. Ota S. Hiregowdara D. Li J. Jiang S. Pasztor L.M. White R.A. Groopman J.E. Avraham H. J. Biol. Chem. 1995; 270: 27742-27751Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 39Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar, 40Sasaki H. Nagura K. Ishino M. Tobioka H. Kotani K. Sasaki T. J. Biol. Chem. 1995; 270: 21206-21219Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). We therefore investigated whether SDF-1α activates RAFTK in L1.2 transfectants. We observed rapid phosphorylation of endogenous murine RAFTK in the transfected L1.2 cells upon SDF-1α stimulation (Fig. 2 A). We also observed an increase in the intrinsic tyrosine kinase activity of RAFTK following SDF-1α treatment, as determined by an autophosphorylation assay and in vitro kinase assay in which poly(Glu:Tyr) (4:1) was used as an exogenous substrate (Fig. 2, B and C). Paxillin and Crk, which are components of focal adhesions, have been shown to play an important role in cell migration and adhesion (41Craig S.W. Johnson R.P. Curr. Opin. Cell Biol. 1996; 8: 74-85Crossref PubMed Scopus (248) Google Scholar, 42Salgia R. Uemura N. Okuda K. Li J.L. Pisick E. Sattler M. de Jong R. Druker B. Heisterkamp N. Chen L.B. Groffen J. Griffin D. J. Biol. Chem. 1995; 270: 29145-29150Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 43Schaller M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (502) Google Scholar). Thus, we sought to investigate whether SDF-1α treatment of CXCR-4 L1.2 cells would result in changes in the phosphorylation state of these proteins. As shown in Fig. 3, A and B, SDF-1α stimulation resulted in enhanced tyrosine phosphorylation of paxillin and Crk. Equivalent amounts of these proteins were present in each lane (bottom panels). It has been shown that upon activation by cytokines the adaptor molecule Crk associates with other components of focal adhesions to enhance signaling (41Craig S.W. Johnson R.P. Curr. Opin. Cell Biol. 1996; 8: 74-85Crossref PubMed Scopus (248) Google Scholar, 42Salgia R. Uemura N. Okuda K. Li J.L. Pisick E. Sattler M. de Jong R. Druker B. Heisterkamp N. Chen L.B. Groffen J. Griffin D. J. Biol. Chem. 1995; 270: 29145-29150Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 43Schaller M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (502) Google Scholar). We therefore investigated whether SDF-1α treatment results in changes in the association of Crk with paxillin and RAFTK. As shown in Fig. 4,A and B, Crk associates with paxillin and RAFTK, and this association was enhanced upon SDF-1α treatment. We investigated the effect of SDF-1α on PI-3 kinase activity. PI-3 kinase is an important mediator of chemotaxis in certain cell types (36Derman M.P. Chen J.Y. Spokes K.C. Songyang Z. Cantley L.G. J. Biol. Chem. 1996; 271: 4251-4255Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 44Thelen M. Uguccioni M. Bosiger J. Biochem. Biophys. Res. Commun. 1995; 217: 1255-1262Crossref PubMed Scopus (90) Google Scholar, 45Kundra V. Escobedo J.A. Kazlauskas A. Kim H.K. Rhee S.G. Williams L.T. Zetter B.R. Nature. 1994; 367: 474-476Crossref PubMed Scopus (399) Google Scholar, 46Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (576) Google Scholar). As shown in Fig. 5 A, SDF-1α treatment increased the PI-3 kinase activity of CXCR-4 L1.2 transfectants. The role of PI-3 kinase in mediating SDF-1α-induced migration was further examined using the selective PI-3 kinase inhibitor, wortmannin. As shown in Fig. 5 B, SDF-1α induced the migration of CXCR-4 L1.2 transfectants, and pretreatment with wortmannin inhibited the SDF-1α-induced migration of cells (Fig. 5 C). Further examination revealed that wortmannin treatment also partially inhibited the SDF-1α-induced tyrosine phosphorylation of paxillin (Fig. 5 D). Equal amounts of paxillin were present in each lane (Fig. 5 D, bottom panel). It has previously been shown that RAFTK acts upstream of MAP kinase and the JNK pathway (39Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar, 47Tokiwa G. Dikic I. Lev S. Schlessinger J. Science. 1996; 273: 792-794Crossref PubMed Scopus (285) Google Scholar). Recently, we have also shown that the β-chemokine, MIP-1β, stimulated JNK kinase in human CCR5 L1.2 transfectants (33Ganju R.K. Dutt P. Wu L. Newman W. Avraham H. Avraham S. Groopman J.E. Blood. 1998; 91: 791-797Crossref PubMed Google Scholar). We further showed that RAFTK mediates activation of JNK in these cells. Fig. 6 Ashows that SDF-1α treatment of CXCR-4 L1.2 cells resulted in the rapid activation of p44/42 MAP kinase. However, no significant effect on JNK or p38 MAP kinase was observed (Fig. 6, B and C). Furthermore MKK-1, which acts upstream of" @default.
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• W2001044562 title "The α-Chemokine, Stromal Cell-derived Factor-1α, Binds to the Transmembrane G-protein-coupled CXCR-4 Receptor and Activates Multiple Signal Transduction Pathways" @default.
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