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• W2014592511 abstract "The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to protein-tyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK·Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK·Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2·FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH2-terminal amino acids 1–401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration. The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to protein-tyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK·Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK·Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2·FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH2-terminal amino acids 1–401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration. Human cytomegalovirus (HCMV) 1The abbreviations used are: HCMVhuman cytomegalovirusSMCsmooth muscle cellMCPmonocyte chemoattractant proteinERKextracellular signal-regulated kinasePTKprotein-tyrosine kinaseFAKfocal adhesion kinaseHAhemagglutininm.o.i.multiplicity of infectionWTwild typeRANTESregulated on activation normal T cell expressed and secretedNTNH2-terminal (1–410 amino acids)PP24-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidineMIPmacrophage inflammatory protein.1The abbreviations used are: HCMVhuman cytomegalovirusSMCsmooth muscle cellMCPmonocyte chemoattractant proteinERKextracellular signal-regulated kinasePTKprotein-tyrosine kinaseFAKfocal adhesion kinaseHAhemagglutininm.o.i.multiplicity of infectionWTwild typeRANTESregulated on activation normal T cell expressed and secretedNTNH2-terminal (1–410 amino acids)PP24-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidineMIPmacrophage inflammatory protein. is a ubiquitous herpesvirus that establishes a life-long latent infection after the primary infection has been cleared. Although anti-viral therapy has appreciably reduced disease in transplant and AIDS patients, HCMV is still a significant problem in congenital disease and bone marrow transplant patients. In addition, HCMV has also been associated with long term diseases such as atherosclerosis, restenosis after angioplasty, chronic rejection after solid organ transplantation, and malignancies (1.Cobbs C.S. Harkins L. Samanta M. Gillespie G.Y. Bharara S. King P.H. Nabors L.B. Cobbs C.G. Britt W.J. Cancer Res. 2002; 62: 3347-3350PubMed Google Scholar, 2.Melnick J.L. Adam E. DeBakery M.E. Infect. Med. 1998; : 479-486Google Scholar, 3.Speir E. Modali R. Huang E.S. Leon M.B. Shawl F. Finkel T. Epstein S.E. Science. 1994; 265: 391-394Crossref PubMed Scopus (701) Google Scholar, 4.Almond P.S. Matas A. Gillingham K. Dunn D.L. Payne W.D. Gores P. Gruessner R. Najarian J.S. Transplant. 1993; 55: 752-757Crossref PubMed Scopus (636) Google Scholar). The development of vascular disease involves a chronic inflammatory process with many contributing factors, and of these, chemokines and their receptors have been identified as key mediators. Interestingly, HCMV encodes a CXC chemokine (UL146), a potential CC chemokine (UL128), and four potential chemokine receptors (US27, US28, UL33, and UL78) with the most characterized being US28 (5.Akter P. Cunningham C. McSharry B.P. Dolan A. Addison C. Dargan D.J. Hassan-Walker A.F. Emery V.C. Griffiths P.D. Wilkinson G.W. Davison A.J. J. Gen. Virol. 2003; 84: 1117-1122Crossref PubMed Scopus (116) Google Scholar, 6.Penfold M.E. Dairaghi D.J. Duke G.M. Saederup N. Mocarski E.S. Kemble G.W. Schall T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9839-9844Crossref PubMed Scopus (303) Google Scholar, 7.Chee M.S. Bankier A.T. Beck S. Bohni R. Browne C.M. Cerny R. Horsnell T. Hutchison III, C.A. Kouzarides T. Martignetti J.A. Preddie E. Satchwell S.C. Tomlinson P. Weston K.M. Barrell B.G. McDougall J.K. Cytomegaloviruses. Springer-Verlag New York Inc., New York1990: 125-171Google Scholar, 8.Chee M.S. Satchwell S.C. Preddie E. Weston K.M. Barrell B.G. Nature. 1990; 344: 774-777Crossref PubMed Scopus (233) Google Scholar). We have previously reported that US28 mediates arterial smooth muscle cell (SMC) migration and that this activity may contribute to viral dissemination and/or acceleration of vascular disease development (9.Streblow D.N. Söderberg-Nauclér C. Vieira J. Smith P. Wakabayashi E. Rutchi F. Mattison K. Altschuler Y. Nelson J.A. Cell. 1999; 99: 511-520Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). human cytomegalovirus smooth muscle cell monocyte chemoattractant protein extracellular signal-regulated kinase protein-tyrosine kinase focal adhesion kinase hemagglutinin multiplicity of infection wild type regulated on activation normal T cell expressed and secreted NH2-terminal (1–410 amino acids) 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine macrophage inflammatory protein. human cytomegalovirus smooth muscle cell monocyte chemoattractant protein extracellular signal-regulated kinase protein-tyrosine kinase focal adhesion kinase hemagglutinin multiplicity of infection wild type regulated on activation normal T cell expressed and secreted NH2-terminal (1–410 amino acids) 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine macrophage inflammatory protein. US28 contains homology to the CC-chemokine receptors (10.Gao J.L. Murphy P.M. J. Biol. Chem. 1994; 269: 28539-28542Abstract Full Text PDF PubMed Google Scholar) and binds to a broad spectrum of chemokines including the CC chemokines RANTES, MCP-1, MCP-3, and MIP-1β and the CX3C chemokine Fractalkine/CX3CL1 (11.Kledal T.N. Rosenkilde M.M. Schwartz T.W. FEBS Lett. 1998; 441: 209-214Crossref PubMed Scopus (169) Google Scholar, 12.Kuhn D.E. Beall C.J. Kolattukudy P.E. Biochem. Biophys. Res. Commun. 1995; 211: 325-330Crossref PubMed Scopus (123) Google Scholar). That CC chemokines fail to compete out Fractalkine binding suggests that Fractalkine binds to additional unique regions of US28 compared with the CC chemokines (11.Kledal T.N. Rosenkilde M.M. Schwartz T.W. FEBS Lett. 1998; 441: 209-214Crossref PubMed Scopus (169) Google Scholar). In 293 cells, RANTES binding to US28 activates ERK1/2 pathways through the G-proteins Gαi1 and Gα16 (13.Billstrom M.A. Johnson G.L. Avdi N.J. Worthen G.S. J. Virol. 1998; 72: 5535-5544Crossref PubMed Google Scholar). We have previously demonstrated that US28-mediated SMC migration also requires chemokine binding by either exogenously added RANTES or endogenously expressed MCP-1 (9.Streblow D.N. Söderberg-Nauclér C. Vieira J. Smith P. Wakabayashi E. Rutchi F. Mattison K. Altschuler Y. Nelson J.A. Cell. 1999; 99: 511-520Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Induction of US28-mediated SMC migration is not blocked by treatment with pertussis toxin, a Gαi/o inhibitor, suggesting that other G-proteins are involved in this event. In fact, we have recently determined that US28 couples with Gα12/13 to signal through RhoA. 2R. Melnychuk, unpublished results.2R. Melnychuk, unpublished results. This activity occurs in a ligand-dependent manner and is required for US28-mediated SMC migration. US28 has also been shown to exhibit constitutive signaling in COS-7 cells and human fibroblasts through both NF-κB and phospholipase C pathways via activation of Gq/11 G-proteins (14.Casarosa P. Bakker R.A. Verzijl D. Navis M. Timmerman H. Leurs R. Smit M.J. J. Biol. Chem. 2001; 276: 1133-1137Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 15.Casarosa P. Menge W.M. Minisini R. Otto C. van Heteren J. Jongejan A. Timmerman H. Moepps B. Kirchhoff F. Mertens T. Smit M.J. Leurs R. J. Biol. Chem. 2003; 278: 5172-5178Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 16.Minisini R. Tulone C. Luske A. Michel D. Mertens T. Gierschik P. Moepps B. J. Virol. 2003; 77: 4489-4501Crossref PubMed Scopus (54) Google Scholar). Interestingly, Fractalkine binding to US28 as well as the US28 inverse agonist (VUF2274) but not CC chemokines antagonized constitutive signaling (15.Casarosa P. Menge W.M. Minisini R. Otto C. van Heteren J. Jongejan A. Timmerman H. Moepps B. Kirchhoff F. Mertens T. Smit M.J. Leurs R. J. Biol. Chem. 2003; 278: 5172-5178Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Sequences have been identified in the US28 cytoplasmic domain that mediate signaling as well as recycling from the plasma membrane (17.Waldhoer M. Casarosa P Rosenkilde M.M Smit M.J. Leurs R Whistler J.L. Schwartz T.W. J. Biol. Chem. 2003; 278: 19473-19482Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Together these data highlight the fact that there are ligand-dependent activities and cell type-specific effects occurring in US28 signaling. Lymphocyte migration induced by ligation of chemokine receptors is generally mediated through the Gαi class of heterotrimeric G-proteins (18.Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar). Upon GPCR stimulation, G-proteins bind directly to the receptor, exchanging GDP for GTP, causing dissociation of the Gα subunit from the Gβγ complex. Downstream effectors of the activated Gα subunit are specific for each type of Gα subunit and can include Ca+2 influx, cAMP, and phosphatidylinositol phosphate. The Gβγ heterodimer activates signaling cascades other than those stimulated by the Gα subunit, which can include the protein-tyrosine kinase (PTK) pathways (19.Inagami T. Eguchi S. Numaguchi K. Motley E.D. Tang H. Matsumoto T. Yamakawa T. J. Am. Soc. Nephrol. 1999; 10: 57-61PubMed Google Scholar). For example ligand binding to CCR2, CCR5, and CXCR1/2 induce cellular migration through the PTK pathway (20.Bacon K.B. Szabo M.C. Yssel H. Bolen J.B. Schall T.J. J. Exp. Med. 1996; 184: 873-882Crossref PubMed Scopus (132) Google Scholar, 21.Mellado M. Rodriguez-Frade J.M. Aragay A. del Real G. Martin A.M. Vila-Coro A.J. Serrano A. Mayor F.J. Martinez A.C. J. Immunol. 1998; 161: 805-813PubMed Google Scholar, 22.Rodriguez-Frade J.M. Vila-Corao A.J. Martin A. Nieto M. Sanchez-Madrid F. Proudfoot A.E.I. Wells T.N. C. Martinez-A C. Mellado M. J. Cell Biol. 1999; 144: 755-765Crossref PubMed Scopus (109) Google Scholar, 23.Feniger-Barish R. Yron I. Meshel T. Matityahu E. Ben-Baruch A. Biochemistry. 2003; 42: 2874-2886Crossref PubMed Scopus (49) Google Scholar). Activation of PTK(s) including that of focal adhesion kinase (FAK) is considered central for cellular migration. The importance of FAK in cellular migration was demonstrated by the inability of fibroblasts isolated from FAK–/– mice to migrate in response to stimuli (24.Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1576) Google Scholar). In addition, overexpression of FAK was shown to enhance movement of CHO cells (25.Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar), and highly invasive tumors characteristically have heightened levels of FAK expression and activity (26.Kornberg L.J. Head Neck. 1998; 20: 745-752Crossref PubMed Scopus (148) Google Scholar, 27.Nurcombe V. Smart C.E. Chipperfield H. Cool S.M. Boilly B. Hondermarck H. J. Biol. Chem. 2000; 275: 30009-30018Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 28.Miyazaki T. Kato H. Nakajima M. Sohda M. Fukai Y. Masuda N. Manda R. Fukuchi M. Tsukada K. Kuwano H. Br. J. Cancer. 2003; 89: 140-145Crossref PubMed Scopus (162) Google Scholar, 29.Schneider G.B. Kurago Z. Zaharias R. Gruman L.M. Schaller M.D. Hendrix M.J. Cancer. 2002; 95: 2508-2515Crossref PubMed Scopus (61) Google Scholar). FAK is comprised of a central kinase domain flanked on one side by an N-terminal FERM (erythrocyte band 4.1-ezrin-radixin-moesin) domain, which is involved in linking FAK to integrins and/or growth factor receptors (30.Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1044) Google Scholar). The focal adhesion targeting domain is located C-terminal of the central kinase domain and is comprised of multiple protein-protein interaction motifs. The binding of paxillin and talin to the FAK-FAT domain facilitates a linkage to the cytoplasmic domain of integrins, which targets FAK to focal adhesions. FAK tyrosine phosphorylation after integrin or growth factor stimulation of cells is enhanced by its association with Src-family PTKs. This leads to the formation of a multiprotein signaling complex in which FAK serves as a scaffold. Mechanistically, after cell stimulation, FAK autophosphorylates at Tyr-397, resulting in the formation of an SH2 docking site. Src-family PTKs bind to FAK at Tyr-397, become activated, and trans-phosphorylate FAK at several other tyrosines including Tyr-925. The SH2 domain of the adaptor protein Grb2 binds to FAK Tyr-925 and forms a signaling complex that includes the nucleotide exchange factor Sos and the small GTP-binding protein Ras. This sequence of events contributes to the subsequent activation of ERK2/mitogen-activated protein kinase. Previously, we have reported that treatment of SMC with the PTK inhibitors genistein or herbimycin A blocked US28-induced cellular migration, suggesting that PTK activity is required for migration of these cells (9.Streblow D.N. Söderberg-Nauclér C. Vieira J. Smith P. Wakabayashi E. Rutchi F. Mattison K. Altschuler Y. Nelson J.A. Cell. 1999; 99: 511-520Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Here we demonstrate that US28 can signal through the non-receptor protein-tyrosine kinases Src and FAK and that this activity is required for the ability of US28 to mediate SMC migration. US28 activation of FAK occurs in a ligand-dependent manner, which is contrary to other reports suggesting that US28 may be constitutively active. In addition, we have determined critical regions of FAK that are required for US28-induced signaling and migration. These findings demonstrate that FAK and Src play integral roles in ligand-dependent, US28-mediated signaling associated with the induction of SMC migration. Cell Lines and Antibodies—The life-extended human pulmonary artery smooth muscle cell line, PAT12 was maintained in Medium 199 supplemented with 20% fetal calf serum and penicillin-streptomycin-l-glutamine (Invitrogen). For migration and in vitro kinase experiments described below PAT1 cells were utilized between passage 5 and 30 post-telomerization. Mouse FAK–/– fibroblasts were maintained on gelatin-coated culture dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin-streptomycin-l-glutamine, nonessential amino acids (Cellgro), and G418 (Sigma; 500 μg/ml) as previously described (24.Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1576) Google Scholar, 31.Sieg D.J. Ilic D. Jones K.C. Damsky C.H. Hunter T. Schlaepfer D.D. EMBO J. 1998; 17: 5933-5947Crossref PubMed Scopus (286) Google Scholar). FAK–/– cells were used in experiments between passage 5 and 15. Anti-Grb2 (C-7), anti-c-Src, anti-phosphotyrosine (PY99), and anti-HA (F-7) monoclonal antibodies were purchased from Santa Cruz Biotechnology. Phospho-specific antibodies to ERK2 (Thr-202/Tyr-204) and total ERK2 were from Cell Signaling Technologies. Paxillin antibodies (5H11) were from Upstate Biotechnologies, and anti-c-Myc tag antibodies were from Covance. The anti-FLAG (M2) monoclonal antibody was from Sigma and the anti-FAK polyclonal serum was previously described (31.Sieg D.J. Ilic D. Jones K.C. Damsky C.H. Hunter T. Schlaepfer D.D. EMBO J. 1998; 17: 5933-5947Crossref PubMed Scopus (286) Google Scholar). FAK in Vitro Kinase Assays—To determine whether US28 promotes the formation of an active FAK kinase complex we performed in vitro kinase assays on immunoprecipitated FAK from human SMC (30.Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1044) Google Scholar, 31.Sieg D.J. Ilic D. Jones K.C. Damsky C.H. Hunter T. Schlaepfer D.D. EMBO J. 1998; 17: 5933-5947Crossref PubMed Scopus (286) Google Scholar). PAT1 SMCs were plated in 10-cm culture dishes and serum-starved for 24 h. The cells were co-infected with Ad-US28 and/or Ad-Trans at multiplicity of infection (m.o.i.) 500. After 16 h the cells were stimulated with RANTES (50 ng/ml) and then harvested at times 0 (unstimulated), 5, 10, and 15 min post-ligand addition. Cells were lysed in radioimmune precipitation assay buffer containing 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, and total FAK was immunoprecipitated using rabbit anti-FAK polyclonal serum and protein A/G-conjugated agarose beads (Santa Cruz). Precipitation reactions were washed 1 time in radioimmune precipitation assay buffer, 2 times in HNTG buffer (50 mm HEPES, 150 mm NaCl, 1% Triton, 10% glycerol, pH 7.4), and 2 times in kinase buffer (20 mm HEPES, 10 mm MgCl2, 10 mm MnCl2, 150 mm NaCl, 10% glycerol, pH 7.4) and then resuspended in kinase buffer plus [γ-32P]ATP. The kinase reaction was allowed to proceed for 15 min at 32 °C and then analyzed by SDS-PAGE and autoradiography. The blots were analyzed by Western blotting for the presence of FAK and Src. Immunoprecipitation Reactions—FAK–/– cells were plated in 10-cm culture dishes and serum-starved for 6 h upon 75% confluence. The cells were co-infected with Ad-Trans and/or Ad-US28 and/or Ad-FAK WT or FAK mutants at m.o.i. 50. After 16 h the cells were stimulated with RANTES (40 ng/ml) and then harvested at times 0 (unstimulated), 5, 10, 15, and 30 min post-ligand addition. Cells were lysed in radioimmune precipitation lysis buffer, total Grb2 was immunoprecipitated, and samples were analyzed by Western blotting using antibodies directed against Tyr(P). Co-precipitation of FAK-HA was demonstrated by stripping the blots in buffer containing 0.1 m Tris, pH 6.8, 1% SDS, and 1% 2-mercaptoethanol and staining using antibodies directed against HA. Before immune-complex reactions, a total of 50 μl of cellular lysate was assayed by SDS-PAGE/Western blotting for the presence of input US28 and FAK using antibodies directed against the HA tag present on both recombinant proteins. US28 co-immunoprecipitation reactions with Src were done as described above except that we used the FLAG-tagged version of US28, and the blots were probed with rabbit polyclonal antibodies directed against c-Src. SMC Migration Assay—Cell migration assays were performed as previously described (9.Streblow D.N. Söderberg-Nauclér C. Vieira J. Smith P. Wakabayashi E. Rutchi F. Mattison K. Altschuler Y. Nelson J.A. Cell. 1999; 99: 511-520Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Briefly, cells were added to the upper well of a Transwell (12-mm diameter, 3.0-μm pore size, Costar Corning, Cambridge, MA) at 1 × 105 cells per well. Cells were serum-starved for 16–24 h. HCMV at m.o.i. 10 was added to the upper well for 2 h. After infection the inserts were washed and transferred to fresh 12-well plates. Cells migrating to the lower chamber were counted at 48–72 h post-infection using a Nikon TE300 microscope at magnification 10×. Experiments were done in at least triplicate wells. Ten random fields were read in each well. The average number of cells per well was determined by multiplying the average number of cells per 10× field by the number of fields per well. Mean and S.D. were calculated. PP2 (1–25 μm; Calbiochem) was added 4 h after infection with HCMV to determine the role of Src in HCMV-US28-mediated SMC migration. For SMC migration studies involving FAK dominant negative adenovirus constructs, SMC were infected with HCMV (m.o.i. 10) for 2 h followed by co-infection with Ad-Trans and Ad-FAK (WT, NT, Phe-397, Arg-454, or Pro-) at m.o.i. of 1000 for an additional 2 h. Subsequently, the Transwell were transferred to fresh 12-well plates. Cellular migration was determined as described above. Recombinant protein levels were monitored by Western blotting and equalized by adjusting the adenoviral vector m.o.i. Adenovirus Construction—Adenoviruses expressing US28-FLAG were previously described (9.Streblow D.N. Söderberg-Nauclér C. Vieira J. Smith P. Wakabayashi E. Rutchi F. Mattison K. Altschuler Y. Nelson J.A. Cell. 1999; 99: 511-520Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Adenovirus vectors expressing US28 with an N-terminal HA tag were constructed by subcloning the DNA fragment (14.Casarosa P. Bakker R.A. Verzijl D. Navis M. Timmerman H. Leurs R. Smit M.J. J. Biol. Chem. 2001; 276: 1133-1137Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) into pAdTet7. This vector contains the tet-responsive enhancer within a minimal CMV promoter followed by the SV40 late poly(A) cassette, adenovirus E1A, and a single loxP site to increase recombination frequency. Recombinant adenoviruses were produced by pAdUS28-HA or pAdFAK (WT, NT, Pro, R454K, Phe-397) construct co-transfection of 293 cells expressing the Cre recombinase with adenovirus DNA (Ad5-ψ5) that contains an E1A/E3-deleted adenovirus genome (32.Hsia D.A. Mitra S.K. Hauck C.R. Streblow D.N. Nelson J.A. Ilic D. Huang S. Li E. Nemerow G.R. Leng J. Spencer K.S. Cheresh D.A. Schlaepfer D.D. J. Cell Biol. 2003; 160: 753-767Crossref PubMed Scopus (454) Google Scholar). Recombinant adenoviruses were expanded on 293-Cre cells, and the bulk stocks were titered on 293 cells by limiting dilution. FAK and US28 expression were driven by co-infection with Ad-Trans expressing the Tet-off transactivator as previously described (9.Streblow D.N. Söderberg-Nauclér C. Vieira J. Smith P. Wakabayashi E. Rutchi F. Mattison K. Altschuler Y. Nelson J.A. Cell. 1999; 99: 511-520Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Immunocytochemistry—FAK–/– fibroblasts were grown in 0.1% gelatin-coated 4-well chamber slides (Nalge-Nunc). US28 and/or FAK was expressed using the adenovirus vectors described above. The cells were washed in phosphate-buffered saline, fixed in phosphate-buffered 1% paraformaldehyde for 10 min at room temperature, permeabilized, and blocked with 0.3% Triton X-100 in phosphate-buffered saline with 10% fetal calf serum and 0.1% sodium azide. Thereafter, the cells were incubated with antibodies against US28-FLAG epitope or FAK-HA epitope or FAK-c-Myc epitope (FAK-NT mutant only) in a 1:200 dilution for 1 h at room temperature. Cells were washed three times in phosphate-buffered saline, and binding of the primary antibody was detected with a fluorescein isothiocyanate tetramethyl-conjugated goat anti-mouse or rhodamine-conjugated goat anti-rabbit antibody for 1 h at room temperature. At this time the cells were also stained for actin using phalloidin (Molecular Probes, Eugene, OR) to monitor alterations in cellular actin cytoskeleton induced by US28 and FAK. Fluorescence-positive cells were visualized on an inverted Nikon fluorescent microscope. US28 Activation of FAK and Src Is Ligand-dependent—We have previously demonstrated that the HCMV-encoded chemokine receptor US28 mediates arterial SMC migration (9.Streblow D.N. Söderberg-Nauclér C. Vieira J. Smith P. Wakabayashi E. Rutchi F. Mattison K. Altschuler Y. Nelson J.A. Cell. 1999; 99: 511-520Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Migration occurred in a ligand-dependent manner that was sensitive to PTK inhibitors, suggesting a role for PTK activation in US28-mediated SMC migration. FAK is a non-receptor PTK with critical importance in facilitating cellular migration. To determine whether FAK plays a role in US28-mediated SMC migration, we first determined whether US28 ligation with RANTES promoted the activation of FAK. For these experiments, we performed in vitro kinase assays on FAK immunoprecipitation reactions. Adenovirus vectors were used to express HCMV-US28 in PAT1 SMC using methods that have been previously described (9.Streblow D.N. Söderberg-Nauclér C. Vieira J. Smith P. Wakabayashi E. Rutchi F. Mattison K. Altschuler Y. Nelson J.A. Cell. 1999; 99: 511-520Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Serum-starved US28-expressing PAT1 cells were untreated (0 time point) or treated with 50 ng/ml RANTES and harvested at 5, 10, or 15 min after the addition of ligand. Total cellular FAK was immunoprecipitated, and the samples were subjected to in vitro kinase assays and examined by SDS-PAGE and autoradiography. RANTES binding to US28 promoted the formation of a FAK·Src kinase complex by 5 min post-addition of ligand, and this activity was maximal at 10 min. FAK·Src kinase activity was reduced by 15 min, suggesting that the complex is transient (Fig. 1). PAT1 cells not expressing US28 failed to promote the FAK·Src kinase complex in the presence of RANTES (data not shown). Untreated US28-expressing PAT1 cells (0 time point) failed to activate the FAK·Src kinase complex, suggesting that this event is not due to an inherent constitutive signaling property of US28 but, rather, requires ligand binding. Western blotting using antibodies directed against FAK showed equal FAK protein levels for each immunoprecipitated sample, and antibodies directed against Src confirmed the association of this protein-tyrosine kinase with FAK (Fig. 1). Interestingly, an additional ∼47-kDa protein was co-immunoprecipitated with the FAK·Src complex, and this protein was highly phosphorylated, suggesting that RANTES binding to US28 facilitates the formation of a FAK and Src kinase complex, which includes additional signaling molecules. US28 Forms a Protein Complex with Src—Previously we demonstrated that US28-mediated SMC migration is blocked by treatment with the pan-PTK inhibitors genistein and herbimycin A (9.Streblow D.N. Söderberg-Nauclér C. Vieira J. Smith P. Wakabayashi E. Rutchi F. Mattison K. Altschuler Y. Nelson J.A. Cell. 1999; 99: 511-520Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Above we show that US28 activates a FAK·Src kinase complex, suggesting that Src plays an important role in US28/FAK signaling. To determine whether Src activity was required for US28-mediated SMC migration, we performed migration assays in the presence of increasing concentrations of PP2. As shown in Fig. 2A, treatment of SMC expressing US28 with PP2 resulted in near total inhibition of cellular migration, which occurred in a dose-dependent manner. We next examined the mechanism involved in US28 activation of Src. We next determined whether US28 complexes with Src by co-immunoprecipitation assays using antibodies directed against the FLAG epitope-tagged version of US28. As shown in Fig. 2B, antibodies directed against US28-FLAG co-precipitated Src upon the addition of RANTES. The complex is formed by 2.5 min after stimulation and is maximal between 2.5 and 5 min. The US28 association with Src returns to near background levels by 10 min (Fig. 2B). The kinetics of Src/US28 association are similar to those observed for Src/β2-adrenergic receptor binding after the addition of ligand (33.Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1234) Google Scholar). These findings suggest that US28 either binds directly to Src or the interaction is mediated through binding to other proteins like β-arrestin, as occurs with the β2-adrenergic receptor. US28 Mediates the Association of FAK with Adaptor Proteins in a Ligand-dependent Manner—Upon cellular activation by extracellular matrix proteins, Src phosphorylates FAK at multiple protein binding sites and increases the ability of FAK to bind adaptor proteins includ" @default.
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• W2014592511 date "2003-12-01" @default.
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• W2014592511 title "Human Cytomegalovirus Chemokine Receptor US28-induced Smooth Muscle Cell Migration Is Mediated by Focal Adhesion Kinase and Src" @default.
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• W2014592511 doi "https://doi.org/10.1074/jbc.m307936200" @default.
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