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β-Chemokine Receptor CCR5 Signals through SHP1, SHP2, and Syk

2000; Elsevier BV; Volume: 275; Issue: 23 Linguagem: Inglês

10.1074/jbc.m000689200

ISSN

1083-351X

Autores

Ramesh K. Ganju, Stephanie A. Brubaker, Rebecca D. Chernock, Shalom Avraham, Jerome E. Groopman,

Tópico(s)

Immunotherapy and Immune Responses

Resumo

The β-chemokine receptor CCR5 has been shown to modulate cell migration, proliferation, and immune functions and to serve as a co-receptor for the human immunodeficiency virus. We and others have shown that CCR5 activates related adhesion focal tyrosine kinase (RAFTK)/Pyk2/CAK-β. In this study, we further characterize the signaling molecules activated by CCR5 upon binding to its cognate ligand, macrophage inflammatory protein-1β (MIP1β). We observed enhanced tyrosine phosphorylation of the phosphatases SHP1 and SHP2 upon MIP1β stimulation of CCR5 L1.2 transfectants and T-cells derived from peripheral blood mononuclear cells. Furthermore, we observed that SHP1 associated with RAFTK. However, using a dominant-negative phosphatase-binding mutant of RAFTK (RAFTKm906), we found that RAFTK does not mediate SHP1 or SHP2 phosphorylation. SHP1 and SHP2 also associated with the adaptor protein Grb2 and the Src-related kinase Syk. Pretreatment of CCR5 L1.2 transfectants or T-cells with the phosphatase inhibitor orthovanadate markedly abolished MIP1β-induced chemotaxis. Syk was also activated upon MIP1β stimulation of CCR5 L1.2 transfectants or T-cells and associated with RAFTK. Overexpression of a dominant-negative Src-binding mutant of RAFTK (RAFTKm402) significantly attenuated Syk activation, whereas overexpression of wild-type RAFTK enhanced Syk activity, indicating that RAFTK acts upstream of CCR5-mediated Syk activation. Taken together, these results suggest that MIP1β stimulation mediated by CCR5 induces the formation of a signaling complex consisting of RAFTK, Syk, SHP1, and Grb2. The β-chemokine receptor CCR5 has been shown to modulate cell migration, proliferation, and immune functions and to serve as a co-receptor for the human immunodeficiency virus. We and others have shown that CCR5 activates related adhesion focal tyrosine kinase (RAFTK)/Pyk2/CAK-β. In this study, we further characterize the signaling molecules activated by CCR5 upon binding to its cognate ligand, macrophage inflammatory protein-1β (MIP1β). We observed enhanced tyrosine phosphorylation of the phosphatases SHP1 and SHP2 upon MIP1β stimulation of CCR5 L1.2 transfectants and T-cells derived from peripheral blood mononuclear cells. Furthermore, we observed that SHP1 associated with RAFTK. However, using a dominant-negative phosphatase-binding mutant of RAFTK (RAFTKm906), we found that RAFTK does not mediate SHP1 or SHP2 phosphorylation. SHP1 and SHP2 also associated with the adaptor protein Grb2 and the Src-related kinase Syk. Pretreatment of CCR5 L1.2 transfectants or T-cells with the phosphatase inhibitor orthovanadate markedly abolished MIP1β-induced chemotaxis. Syk was also activated upon MIP1β stimulation of CCR5 L1.2 transfectants or T-cells and associated with RAFTK. Overexpression of a dominant-negative Src-binding mutant of RAFTK (RAFTKm402) significantly attenuated Syk activation, whereas overexpression of wild-type RAFTK enhanced Syk activity, indicating that RAFTK acts upstream of CCR5-mediated Syk activation. Taken together, these results suggest that MIP1β stimulation mediated by CCR5 induces the formation of a signaling complex consisting of RAFTK, Syk, SHP1, and Grb2. human immunodeficiency virus macrophage inflammatory protein regulated on activation normal T-cell expressed related adhesion focal tyrosine kinase wild-type RAFTK fluorescence-activated cell sorter glutathioneS-transferase Chemokines and their receptors play important roles in cell migration, growth, and host inflammatory responses (1.Premack B.A. Schall T.J. Nat. Med. 1996; 2: 1174-1178Crossref PubMed Scopus (572) Google Scholar, 2.Luster A.D. N. Engl. J. Med. 1998; 338: 436-445Crossref PubMed Scopus (3240) Google Scholar, 3.Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar). These receptors also serve as co-receptors for the human immunodeficiency virus (HIV)1 (4.Littman D.R. Cell. 1998; 93: 677-680Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 5.Moore J.P. Trkola A. Dragic T. Curr. Opin. Immunol. 1997; 9: 551-562Crossref PubMed Scopus (451) Google Scholar, 6.Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3624) Google Scholar). Recently, chemokines also have been implicated in the development of Kaposi's sarcoma via Kaposi's sarcoma herpes virus (human herpes virus (HHV-8)), which encodes for several functional homologues of certain chemokines like MIP and of α-chemokine receptors, including CXCR1, CXCR2, and CXCR3 (7.Nicholas J. Ruvolo V.R. Burns W.H. Sandford G. Wan X. Ciufo D. Hendrickson S.B. Guo H.G. Hayward G.S. Reitz M.S. Nat. Med. 1997; 3: 287-292Crossref PubMed Scopus (337) Google Scholar, 8.Murphy P.M. 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Berger E.A. Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2438) Google Scholar). CCR5 is activated by the β-chemokines MIP1β, MIP1α, and RANTES. MIP1β has higher specificity for CCR5 than do MIP1α and RANTES, which also bind to CCR1 and CCR3 (1.Premack B.A. Schall T.J. Nat. Med. 1996; 2: 1174-1178Crossref PubMed Scopus (572) Google Scholar, 3.Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, 12.Alkhatib G. Combadiere C. Broder C.C. Feng Y. Kennedy P.E. Murphy P.M. Berger E.A. Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2438) Google Scholar). Knockout mice lacking CCR5 revealed partial defects in macrophages and also showed enhanced T-cell-dependent immune response and delayed type hypersensitivity reaction, suggesting that CCR5 may play an important role in down-regulating T-cell-dependent immune responses (13.Zhou Y. Kurihara T. Ryseck R.P. Yang Y. Ryan C. Loy J. Warr G. Bravo R. J. Immunol. 1998; 160: 4018-4025PubMed Google Scholar). Despite the emerging role of CCR5 and its ligands in HIV infection and the immune response, relatively little is known about the signaling pathways mediated by this receptor. CCR5 has been shown to induce calcium signals and chemotaxis upon binding to the macrophage-tropic HIV gp160 recombinant envelope protein (14.Weissman D. Rabin R.L. Arthos J. Rubbert A. Dybul M. Swofford R. Venkatesan S. Farber J.M. Fauci A.S. Nature. 1997; 389: 981-985Crossref PubMed Scopus (310) Google Scholar). We (15.Ganju R.K. Dutt P. Wu L. Newman W. Avraham H. Avraham S. Groopman J.E. Blood. 1998; 91: 791-797Crossref PubMed Google Scholar) and others (16.Davis 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) have also recently shown that CCR5, upon binding to its cognate ligand (MIP1β) or to the HIV-1 envelope glycoprotein from a macrophage-tropic strain, activates a member of the focal adhesion kinase family called related adhesion focal tyrosine kinase (RAFTK; also known as Pyk2 and CAK-β). We further demonstrated that RAFTK associates with the cytoskeletal protein paxillin upon CCR5 activation (15.Ganju R.K. Dutt P. Wu L. Newman W. Avraham H. Avraham S. Groopman J.E. Blood. 1998; 91: 791-797Crossref PubMed Google Scholar). To further elucidate MIP1β-induced CCR5 signaling pathways, we have delineated the roles of the SH2 domain-containing cytoplasmic tyrosine phosphatase subfamily members SHP1 and SHP2 and of the Src-related kinase Syk. SHP1 (also named SHPTP1, PTPK, HC, or SHP) is predominantly expressed in hematopoietic cells, whereas SHP2 (also called SHPTP2, PTP1D, SYP, PTP2C, or SHPTP3) is expressed ubiquitously (17.Streuli M. Curr. Opin. Cell Biol. 1996; 8: 182-188Crossref PubMed Scopus (165) Google Scholar, 18.Neel B.G. Curr. Opin. Immunol. 1997; 9: 405-420Crossref PubMed Scopus (140) Google Scholar). SHP1 acts as a negative regulator of intracellular signaling (19.Haque S.J. Harbor P. Tabrizi M. Yi T. Williams B.R. J. Biol. Chem. 1998; 273: 33893-33896Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 20.Dong Q. Siminovitch K.A. Fialkow L. Fukushima T. Downey G.P. J. Immunol. 1999; 162: 3220-3230PubMed Google Scholar). In contrast, SHP2 appears to play a positive role in regulating various growth factor-induced signaling pathways (21.Frearson J.A. Alexander D.R. J. Exp. Med. 1998; 187: 1417-1426Crossref PubMed Scopus (101) Google Scholar, 22.Nakamura K. Cambier J.C. J. 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Commun. 1997; 238: 261-266Crossref PubMed Scopus (43) Google Scholar). Mice deficient in SHP1 (motheaten or viable motheaten) suffer from several immunological, inflammatory, and hematological abnormalities (29.Shultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 30.Shultz L.D. Rajan T.V. Greiner D.L. Trends Biotechnol. 1997; 15: 302-307Abstract Full Text PDF PubMed Scopus (146) Google Scholar), whereas SHP2 mutant mice possess multiple defects in mesodermal patterning and die at mid-gestation (31.Qu C.K., Yu, W.M. Azzarelli B. Cooper S. Broxmeyer H.E. Feng G.S. Mol. Cell. Biol. 1998; 18: 6075-6082Crossref PubMed Scopus (107) Google Scholar). Furthermore, recently, it was shown that chemotactic responses to chemokine stromal cell-derived factor-1α were altered in mature and immature hematopoietic cells derived frommotheaten mice (32.Kim C.H. Qu C.K. Hangoc G. Cooper S. Anzai N. Feng G.S. Broxmeyer H.E. J. Exp. Med. 1999; 190: 681-690Crossref PubMed Scopus (81) Google Scholar). Syk, a cytoplasmic protein-tyrosine kinase, plays an important role in the signaling pathways mediated by the B-cell antigen, Fc receptor, T-cell antigen receptor, and integrin receptor and thereby modulates cell growth and chemotaxis (33.Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (386) Google Scholar, 34.Latour S. Fournel M. Veillette A. Mol. Cell. Biol. 1997; 17: 4434-4441Crossref PubMed Scopus (68) Google Scholar, 35.Gao J. Zoller K.E. Ginsberg M.H. Brugge J.S. Shattil S.J. EMBO J. 1997; 16: 6414-6425Crossref PubMed Scopus (160) Google Scholar, 36.Brumbaugh K.M. Binstadt B.A. Billadeau D.D. Schoon R.A. Dick C.J. Ten R.M. Leibson P.J. J. Exp. Med. 1997; 186: 1965-1974Crossref PubMed Scopus (120) Google Scholar, 37.Turner M. Gulbranson-Judge A. Quinn M.E. Walters A.E. MacLennan I.C. Tybulewicz V.L. J. Exp. Med. 1997; 186: 2013-2021Crossref PubMed Scopus (143) Google Scholar). Syk interacts with various tyrosine-phosphorylated proteins (38.Yanaga F. Poole A. Asselin J. Blake R. Schieven G.L. Clark E.A. Law C.L. Watson S.P. Biochem. J. 1995; 311: 471-478Crossref PubMed Scopus (131) Google Scholar, 39.Sidorenko S.P. Law C.L. Chandran K.A. Clark E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 359-363Crossref PubMed Scopus (62) Google Scholar). Prior analysis of RAFTK indicated that tyrosine 402 in its N-terminal domain binds to the SH2 domain of Src kinases and activates such Src kinases upon lipopolysaccharide or bradykinin treatment (40.Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (877) Google Scholar). This study demonstrates that MIP1β stimulation of CCR5 transfectants induces the tyrosine phosphorylation of SHP1 and SHP2 and activates Syk. This induction also results in the formation of a signaling complex consisting of RAFTK, Syk, SHP1, and Grb2. These results indicate that RAFTK acts upstream of Syk and suggest that it does not regulate the SHP1 and SHP2 phosphorylation mediated by CCR5. Anti-RAFTK antibodies were generated as described previously (41.Li J. Avraham H. Rogers R.A. Raja S. Avraham S. Blood. 1996; 88: 417-428Crossref PubMed Google Scholar). This antiserum recognized both human and murine forms of RAFTK and did not cross-react with focal adhesion kinase. Antibodies to Syk, SHP1, and SHP2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-phosphotyrosine antibody (4G10) was a generous gift from Dr. Brian Druker (Oregon Health Sciences University, Portland, OR). Electrophoresis reagents and nitrocellulose membranes were obtained from Bio-Rad. The protease inhibitors leupeptin and α1-antitrypsin and all other reagents were acquired from Sigma. Indo-1/AM was purchased from Molecular Probes, Inc. (Eugene, OR). CCR5 was transfected into the pre-B lymphoma cell line L1.2 as described previously (42.Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffing N. Borsetti A. Cardoso A.A. Desjardin E. Newman W. Gerard C. Sodroski J. Nature. 1996; 384: 179-183Crossref PubMed Scopus (1081) Google Scholar, 43.Wu L. Paxton W.A. Kassam N. Ruffing N. Rottman J.B. Sullivan N. Choe H. Sodroski J. Newman W. Koup R.A. Mackay C.R. J. Exp. Med. 1997; 185: 1681-1691Crossref PubMed Scopus (639) Google Scholar), and transfectants were selected in medium containing mycophenolic acid. FACS analysis was used to monitor the cell-surface expression of CCR5. CCR5 was expressed at a high level in these cells (42.Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffing N. Borsetti A. Cardoso A.A. Desjardin E. Newman W. Gerard C. Sodroski J. Nature. 1996; 384: 179-183Crossref PubMed Scopus (1081) Google Scholar). The β-chemokines MIP1α, MIP1β, and RANTES bind with high affinity to the expressed CCR5 receptors. These cells possess properties characteristic of native CCR5, as they bind to macrophage-tropic HIV-1 gp120 in the presence of soluble human CD4 (42.Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffing N. Borsetti A. Cardoso A.A. Desjardin E. Newman W. Gerard C. Sodroski J. Nature. 1996; 384: 179-183Crossref PubMed Scopus (1081) Google Scholar). CCR5 transfectants and mutants were grown in RPMI 1640 medium (containing 10% fetal calf serum, 2 mmol/liter glutamine, 1 mmol/liter sodium pyruvate, 50 μg/ml penicillin, 50 μg/ml streptomycin, and 55 μmol/liter β-mercaptoethanol) supplemented with 100 nmol/liter sodium hypoxanthine, 16 nmol/liter thymidine, 2.5 μg/ml mycophenolic acid, and 125 μg/ml xanthine. CCR5 L1.2 transfectants containing RAFTK constructs were grown in mycophenolic acid medium containing 0.8 mg/liter Geneticin (G418, Life Technologies, Inc.). Transfectants of mutants RAFTKm402 and RAFTKm906 and wild-type RAFTK (RAFTKWT) were produced by transfection of the CCR5 L1.2 cells with the RAFTKm402, RAFTKm906, or RAFTKWT construct, respectively (15.Ganju R.K. Dutt P. Wu L. Newman W. Avraham H. Avraham S. Groopman J.E. Blood. 1998; 91: 791-797Crossref PubMed Google Scholar). pcDNA vector without an RAFTK construct was used as a control. Mutants RAFTKm906 and RAFTKm402 were generated by replacing Tyr906 and Tyr402 with Phe, respectively, by site-directed mutagenesis. Tyr906 of RAFTK is the putative binding site for phosphatases, and Tyr402has previously been shown to bind to Src kinases (40.Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (877) Google Scholar). Plasmids carrying RAFTKm906, RAFTKm402, RAFTKWT, or pcDNA control vector were transfected by electroporation into the CCR5 L1.2 cells using Bio-Rad electroporation equipment. The transfectants were selected in medium containing mycophenolic acid and G418. The double mutants expressed equal amounts of CCR5 as determined by FACS analysis. Several clones of double transfectants were used in the signaling studies. T-cell-enriched, monocyte-depleted cultures were generated from peripheral blood mononuclear cells as described (15.Ganju R.K. Dutt P. Wu L. Newman W. Avraham H. Avraham S. Groopman J.E. Blood. 1998; 91: 791-797Crossref PubMed Google Scholar, 44.Alfano M. Schmidtmayerova H. Amella C.A. Pushkarsky T. Bukrinsky M. J. Exp. Med. 1999; 190: 597-606Crossref PubMed Scopus (96) Google Scholar). Briefly, peripheral blood mononuclear cells were separated by Ficoll-Hypaque gradient centrifugation and two rounds of adherence to plastic. Non-adherent cells were stimulated with phytohemagglutinin (5 μg/ml) for 3 days. Cells were removed to fresh medium supplemented with recombinant human interleukin-2 (Advanced Biotechnologies, Columbia, MD). Three-week-old activated T-cells, which were found to be ∼35% positive for CCR5 by FACS analysis, were used for further studies. Cells were washed twice with 1× Hanks' buffered salt solution (Sigma), resuspended at 10 × 106 cells/ml in 1× Hanks' buffered salt solution, and then starved of serum for 2 h at 37 °C. The serum-starved cells were stimulated with 200 ng/ml MIP1β at 37 °C for various time periods. Following stimulation, cells were lysed using modified radioimmune precipitation assay buffer (50 mmol/liter Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mmol/liter NaCl, 1 mmol/liter phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 mmol/liter sodium vanadate, 10 mmol/liter sodium fluoride, and 10 mmol/liter sodium pyrophosphate). Total cell lysates were clarified by centrifugation at 10,000 ×g for 10 min. Protein concentrations were determined by protein assay (Bio-Rad). Immunoprecipitation studies were conducted as described (45.Ganju R.K. Brubaker S.A. Meyer J. Dutt P. Yang Y. Qin S. Newman W. Groopman J.E. J. Biol. Chem. 1998; 273: 23169-23175Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). Briefly, identical amounts of protein from each time point were clarified by incubation with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) for 1 h at 4 °C. Protein A-Sepharose was removed by brief centrifugation, and the supernatants were incubated with different primary antibodies as described below for each experiment for 2 h at 4 °C. Immunoprecipitation of the antibody-antigen complexes was performed by incubation at 4 °C overnight with 50 μl of protein A-Sepharose (10% suspension). Nonspecific bound proteins were removed by washing the Sepharose beads three times with modified radioimmune precipitation assay buffer and one time with phosphate-buffered saline. Immune complexes were solubilized in 30 μl of 2× Laemmli buffer, and samples were separated on 8 or 12% SDS-polyacrylamide gel and then transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat milk protein for 1 h and probed with primary antibody for 3 h at room temperature or at 4 °C overnight. Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech). The densitometric scanning of films was done using a Bio-Rad Model G5–700 imaging densitometer. The Syk kinase assay was performed by first immunoprecipitating lysates with anti-Syk antibody. The immune complexes were then washed twice with radioimmune precipitation assay buffer and twice with Syk kinase buffer (20 mm Hepes, 50 mm NaCl, 10 μmNa3VO4, 5 mm MgCl2, and 5 mm MnCl2). The complex was incubated in Syk kinase buffer and 5 μCi of [γ-32P]ATP for 20 min at 30 °C. The reaction was terminated by adding 4× SDS sample buffer and boiling the samples for 5 min at 100 °C. Proteins were then separated on 8% SDS-polyacrylamide gel and detected by autoradiography. Rabbit IgG was used as a negative control. The RAFTK C-terminal domain (amino acids 681–1009)-glutathione S-transferase (GST) fusion protein was produced as described (46.Ganju 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). Briefly, the fusion protein was amplified by polymerase chain reaction and cloned into the pGEX-2T expression vector (Amersham Pharmacia Biotech). The GST fusion protein was produced by 1 mmisopropyl-β-d-thiogalactopyranoside induction and purified by affinity chromatography on a glutathione-Sepharose column (Amersham Pharmacia Biotech) according to the manufacturer's recommendations. Grb2-GST fusion proteins were purchased from Santa Cruz Biotechnology. For the binding experiments, 5 μg of GST fusion protein were mixed with 1 mg of cell lysate and incubated for 1 h at 4 °C on a rotatory shaker. GST protein (Santa Cruz Biotechnology) was used as a control. The complex was pre-absorbed by adding 50 μl of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). The samples were incubated overnight at 4 °C on a rotatory shaker. The beads were centrifuged and washed three times with modified radioimmune precipitation assay buffer and once with 1× phosphate-buffered saline. The bound proteins were eluted by boiling in Laemmli sample buffer and subjected to 8% SDS-polyacrylamide gel electrophoresis and Western blot analysis. The assay was performed in 24-well plates containing 5-μm porosity inserts (Costar Corp., Cambridge, MA) as described (45.Ganju R.K. Brubaker S.A. Meyer J. Dutt P. Yang Y. Qin S. Newman W. Groopman J.E. J. Biol. Chem. 1998; 273: 23169-23175Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). Briefly, cells were resuspended at 6.6 × 106/ml in RPMI 1640 medium containing 2.5% bovine serum albumin. 50 ng of MIP1β were added to the bottom wells, and 150 μl of cells (1 × 106) untreated or pretreated with different concentrations of sodium orthovanadate were loaded onto the inserts. Cells migrating to the bottom wells were collected, centrifuged, and counted. The results shown are representative of findings from three independent experiments. Cytoplasmic tyrosine phosphatases have been shown to be important positive and negative regulators of signaling pathways. To characterize the role that phosphatases play in CCR5-mediated signal transduction pathways, CCR5 transfectants were stimulated with MIP1β, and lysates were analyzed for SHP1 and SHP2 tyrosine phosphorylation. As shown in Fig. 1 (A andC, respectively), MIP1β stimulation of CCR5 transfectants resulted in an increase in tyrosine phosphorylation of SHP1 and SHP2. Equal amounts of SHP1 and SHP2 proteins were present in each lane (Fig.1, A and C, lower panels). We also found that MIP1β stimulation induced a slight increase in tyrosine phosphorylation of SHP1 and SHP2 (Fig. 1, B andD, respectively) in activated T-cells derived from peripheral blood mononuclear cells. These T-cells were found to be ∼35% positive for CCR5 (data not shown). The reduced SHP1 and SHP2 phosphorylation in T-cells as compared with CCR5 L1.2 cells may be due to differences in the number of CCR5 receptors on these cells and/or to differences between the cell types. In addition, RAFTK has also been shown to have lower tyrosine phosphorylation in MIP1β-stimulated T-cells as compared with the similarly stimulated CCR5 L1.2 transfectants (15.Ganju R.K. Dutt P. Wu L. Newman W. Avraham H. Avraham S. Groopman J.E. Blood. 1998; 91: 791-797Crossref PubMed Google Scholar). Tyr906 of RAFTK may act as a putative binding site for SHP proteins. Therefore, we examined the association of RAFTK with SHP1. As shown in Fig.2 A, SHP1 associated with RAFTK constitutively; this association was modestly increased upon MIP1β stimulation. To further confirm this association, we used a GST fusion protein containing the C-terminal domain of RAFTK. As shown in Fig.2 B, SHP1 was observed to associate with this fusion protein. SHP1 also associated with Grb2 upon MIP1β stimulation (Fig.2 C). Since SHP2 was also tyrosine-phosphorylated following MIP1β stimulation, we studied whether it associated with the adaptor protein Grb2. As shown in Fig. 2 D, SHP2 associated with Grb2, and this association increased upon chemokine stimulation. Since SHP1 was shown to associate with RAFTK upon MIP1β stimulation, we were interested in whether RAFTK modulated the MIP1β-stimulated phosphorylation of SHP1. To address this question, we created double-transfected L1.2 cells that expressed human CCR5 and RAFTKm906, in which Tyr906 was replaced with Phe. Tyr906 of RAFTK is a putative binding site for phosphatases. As shown in Fig.3 (A and B, respectively), there was no significant increase in tyrosine phosphorylation of SHP1 and SHP2 in the RAFTKm906transfectants as compared with the pcDNA transfectants. An equal amount of SHP1 and SHP2 proteins was present in all samples (Fig. 3,A and B, lower panels). Tyrosine phosphatases have been shown to play an important role in chemotaxis. To determine the potential role of phosphatases in MIP1β-stimulated chemotaxis, we examined MIP1β-induced migration in the presence of different concentrations of the phosphatase inhibitor orthovanadate. As shown in Fig.4 (A and B, respectively), orthovanadate treatment attenuated the MIP1β-induced migration of CCR5 L1.2 cells and activated T-cells in a dose-dependent manner. 100 μm orthovanadate resulted in ∼85% inhibition of CCR5 L1.2 and ∼70% inhibition of T-cell chemotaxis. No effect on cell survival or growth was observed under similar conditions. The Src-related kinase Syk has been shown to participate in various signaling pathways regulating cell growth and adhesion. To characterize the role of Syk in CCR5-mediated signal transduction pathways, CCR5 transfectants or T-cells were stimulated with MIP1β, and the lysates were analyzed for Syk kinase activation. As shown in Fig. 5 A(upper panel), MIP1β stimulation of the CCR5 L1.2 transfectants resulted in enhanced tyrosine phosphorylation of Syk. Equal amounts of Syk protein were present in each lane (Fig.5 A, lower panel). In addition, MIP1β stimulation induced Syk-autophosphorylating activity in CCR5 L1.2 transfectants (Fig. 5 B) and, to a lesser degree, in T-cells (Fig. 5 C). Similar to SHP1 and SHP2 phosphorylation, Syk-autophosphorylating activity was reduced in T-cells as compared with the CCR5 L1.2 transfectants upon MIP1β stimulation. To further characterize the role that Syk might play in CCR5-mediated signaling, we sought to identify proteins that associate with Syk upon MIP1β stimulation. We observed a constitutive association of Syk with RAFTK, an association that was enhanced upon MIP1β stimulation (Fig.6 A). RAFTK has previously been shown to be phosphorylated and activated by MIP1β (15.Ganju R.K. Dutt P. Wu L. Newman W. Avraham H. Avraham S. Groopman J.E. Blood. 1998; 91: 791-797Crossref PubMed Google Scholar). Furthermore, Syk was also shown to associate with the SH2 domain of Grb2. This association was enhanced upon MIP1β stimulation (Fig. 6 B). Since we observed that Syk was activated and associated with RAFTK upon MIP1β stimulation, we subsequently studied the association of Syk with SHP1. As shown, Syk also associated with SHP1 (Fig.7), and this association was enhanced upon MIP1β stimulation.Figure 7SHP1 associates with Syk. Lysates from CCR5 L1.2 cells stimulated with MIP1β for different time periods as indicated were immunoprecipitated (IP) with SHP1 and run on SDS-polyacrylamide gel. The blots were subjected to serial blotting with Syk (upper panel), followed by blotting with SHP1 (lower panel). The C lanerepresents immunoprecipitates with control IgG. WB, Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since RAFTK associated with Syk, we wanted to see whether this association was important for Syk activation. To address this question, we created double-transfected L1.2 cells that expressed human CCR5 and RAFTKm402 (which lacks the Src-binding site) or CCR5 and RAFTKWT. Up to a 3-fold increase in Syk activation was observed in the MIP1β-treated CCR5-RAFTKWT cells as compared with the pcDNA-transfected cells (Fig.8 A). We also observed an ∼2-fold decrease in Syk activity in the CCR5 L1.2 transfectants overexpressing the dominant-negative mutant RAFTKm402 as compared with transfectants expressing the pcDNA vector (Fig.8 B). These studies suggest that RAFTK may regulate Syk kinase activation in these cells. In our earlier studies using CCR5-transfected murine pre-B lymphoma L1.2 cells as a model, we have shown that MIP1β stimulation results in activation of RAFTK/Pyk2 (15.Ganju R.K. Dutt P. Wu L. Newman W. Avraham H. Avraham S. Groopman J.E. Blood. 1998; 91: 791-797Crossref PubMed Google Scholar). Furthermore, HIV gp120 binding to CCR5 and chemokine stimulation have also been shown to induce RAFTK/Pyk2 phosphorylation (16.Davis 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, 47.Dikic I. Schlessinger J. J. Biol. 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A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar). Recent studies of hematopoietic cells from motheaten mice indicate that SHP1 plays an important role in the regulation of the stromal cell-derived factor-1-induced signaling pathway (32.Kim C.H. Qu C.K. Hangoc G. Cooper S. Anzai N. Feng G.S. Broxmeyer H.E. J. Exp. Med. 1999; 190: 681-690Crossref PubMed Scopus (81) Google Scholar). We have observed that SHP1 is associated with RAFTK. However, RAFTK does not appear to mediate the MIP1β-stimulated phosphorylation of SHP1 or SHP2 since overexpression of a dominant-negative phosphatase-binding mutant of RAFTK had little effect on MIP1β-stimulated SHP1 or SHP2 phosphorylation. We also observed that chemokine stimulation resulted in the enhanced association of SHP1 and SHP2 with Syk and Grb2. In different signaling pathways, SHP1 and SHP2 are known to associate with various signaling molecules, including Vav, Grb2, SOS, and SLP-76 via their SH2 domains (53.Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky S. Feldman R.A. Cantley L.C. Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (831) Google Scholar, 54.Veillette A. Thibaudeau E. Latour S. J. Biol. Chem. 1998; 273: 22719-22728Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 55.Fujioka Y. Matozaki T. Noguchi T. Iwamatsu A. Yamao T. Takahashi N. Tsuda M. Takada T. Kasuga M. Mol. Cell. Biol. 1996; 16: 6887-6899Crossref PubMed Scopus (382) Google Scholar, 56.Zhang S. Broxmeyer H.E. Biochem. Biophys. Res. Commun. 1999; 254: 440-445Crossref PubMed Scopus (72) Google Scholar). In addition to their role as phosphatases, SHP1 and SHP2 may act as adaptor proteins by providing docking sites for the recruitment of downstream signaling molecules. The present study indicates that tyrosine phosphatases may play an important role in the regulation of MIP1β-induced migration, as orthovanadate treatment markedly attenuated MIP1β-stimulated chemotaxis. Recently, platelet-derived growth factor-induced migration was shown to be regulated by SHP2 (48.Qi J.H. Ito N. Claesson-Welsh L. J. Biol. Chem. 1999; 274: 14455-14463Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). MIP1β stimulation also induced activation of Syk, a Src-related kinase. Syk has been shown to participate in signal transduction pathways mediated by B- and T-cell antigen receptors, the Fc receptor, various growth factor receptors, and integrin receptors (33.Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (386) Google Scholar, 34.Latour S. Fournel M. Veillette A. Mol. Cell. Biol. 1997; 17: 4434-4441Crossref PubMed Scopus (68) Google Scholar, 35.Gao J. Zoller K.E. Ginsberg M.H. Brugge J.S. Shattil S.J. EMBO J. 1997; 16: 6414-6425Crossref PubMed Scopus (160) Google Scholar, 36.Brumbaugh K.M. Binstadt B.A. Billadeau D.D. Schoon R.A. Dick C.J. Ten R.M. Leibson P.J. J. Exp. Med. 1997; 186: 1965-1974Crossref PubMed Scopus (120) Google Scholar, 37.Turner M. Gulbranson-Judge A. Quinn M.E. Walters A.E. MacLennan I.C. Tybulewicz V.L. J. Exp. Med. 1997; 186: 2013-2021Crossref PubMed Scopus (143) Google Scholar). However, the G-protein-coupled m1 muscarinic receptor does not activate Syk. Tyr402 (autophosphorylation site) of RAFTK has been shown to bind to Src kinases, which results in their activation (40.Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (877) Google Scholar). In the present study, we observed that Syk associated with RAFTK and that this association was enhanced upon MIP1β stimulation. RAFTK association with another Src-related kinase, Fyn, has been shown to play an important role in mediating T-cell receptor signal transduction (46.Ganju 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, 57.Qian D. Lev S. van Oers N.S. Dikic I. Schlessinger J. Weiss A. J. Exp. Med. 1997; 185: 1253-1259Crossref PubMed Scopus (150) Google Scholar). Furthermore, RANTES has been shown to induce ZAP-70 activity and its association with focal adhesion kinase in T-cells (58.Bacon 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). In this study, RAFTK appeared to partially mediate Syk activation, as overexpression of a Src-binding mutant of RAFTK resulted in the reduced activation of Syk, whereas overexpression of wild-type RAFTK enhanced Syk activity. Recently, Syk was shown to be upstream of RAFTK/Pyk2 phosphorylation in Fcε receptor-1-induced tyrosine phosphorylation in mast cells, whereas Pyk2 phosphorylation by thrombin and the adenosine G-protein-coupled receptor was independent of RAFTK/Pyk2 in these cells (59.Okazaki H. Zhang J. Hamawy M.M. Siraganian R.P. J. Biol. Chem. 1997; 272: 32443-32447Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We also observed that Syk associated with SHP1 and that this association was enhanced by chemokine stimulation. Tyrosine phosphatases can cause activation of Src kinases (60.Somani A.K. Bignon J.S. Mills G.B. Siminovitch K.A. Branch D.R. J. Biol. Chem. 1997; 272: 21113-21119Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). SHP1 has been shown to regulate Syk activity, as overexpression of SHP1-inactive mutants in B lymphoma cell lines results in enhanced Syk kinase activity (61.Dustin L.B. Plas D.R. Wong J. Hu Y.T. Soto C. Chan A.C. Thomas M.L. J. Immunol. 1999; 162: 2717-2724PubMed Google Scholar). Taken together, our results provide new information regarding various downstream signaling molecules involved in CCR5-mediated signaling pathways. We have found that MIP1β stimulation of CCR5 activates Syk and increases the tyrosine phosphorylation of the SH2-domain containing phosphatases SHP1 and SHP2. This results in the formation of a multimeric complex consisting of RAFTK, Syk, Grb2, and SHP1. RAFTK was shown to partially mediate the activation of Syk, but had no significant effect on SHP1 or SHP2 tyrosine phosphorylation. These results suggest that RAFTK differentially regulates several downstream signaling targets that are activated upon CCR5 stimulation. We are grateful to Walter Newman and Lijun Wu (LeukoSite, Inc.) for providing the CCR5 L1.2 transfectants. We also thank Janet Delahanty for editing, Nancy DesRosiers and Dan Kelley for preparation of the figures, and Simone Jadusingh for typing the manuscript.

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