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• W1969732387 abstract "Janus kinases Jak1 and Tyk2 play an important role in urokinase-type plasminogen activator (uPA)-dependent signaling. We have recently demonstrated that both kinases are associated with the uPA receptor (uPAR) and mediate uPA-induced activation of signal transducers and activators of transcription (Stat1, Stat2, and Stat4) in human vascular smooth muscle cells (VSMC). Janus kinases are not only required for Stat activation but may also interfere with other intracellular signaling pathways. Here we report that in VSMC, Tyk2 interacts with a downstream signaling cascade involving phosphatidylinositol 3-kinase (PI3-K). We demonstrate that uPA induces PI3-K activation, which is abolished in VSMC expressing the dominant negative form of Tyk2. The regulatory subunit p85 of PI3-K co-immunoprecipitates with Tyk2 but not with Jak1, Jak2, or Jak3, and uPA stimulation increases the PI3-K activity in Tyk2 immunoprecipitates. Tyk2 directly binds to either of the two Src homology 2(SH2)p85 domains in a uPA-dependent fashion. We provide evidence that the Tyk2-mediated PI3-K activation in response to uPA is required for VSMC migration. Thus, two unrelated structurally distinct specific inhibitors of PI3-K, wortmannin and LY294002, prevent VSMC migration induced by uPA. No migratory effect of uPA was observed in VSMC expressing the dominant negative form of Tyk2. Our results underscore the versatile function of Tyk2 in uPA-related intracellular signaling and indicate that PI3-K plays a selective role in the regulation of VSMC migration. Janus kinases Jak1 and Tyk2 play an important role in urokinase-type plasminogen activator (uPA)-dependent signaling. We have recently demonstrated that both kinases are associated with the uPA receptor (uPAR) and mediate uPA-induced activation of signal transducers and activators of transcription (Stat1, Stat2, and Stat4) in human vascular smooth muscle cells (VSMC). Janus kinases are not only required for Stat activation but may also interfere with other intracellular signaling pathways. Here we report that in VSMC, Tyk2 interacts with a downstream signaling cascade involving phosphatidylinositol 3-kinase (PI3-K). We demonstrate that uPA induces PI3-K activation, which is abolished in VSMC expressing the dominant negative form of Tyk2. The regulatory subunit p85 of PI3-K co-immunoprecipitates with Tyk2 but not with Jak1, Jak2, or Jak3, and uPA stimulation increases the PI3-K activity in Tyk2 immunoprecipitates. Tyk2 directly binds to either of the two Src homology 2(SH2)p85 domains in a uPA-dependent fashion. We provide evidence that the Tyk2-mediated PI3-K activation in response to uPA is required for VSMC migration. Thus, two unrelated structurally distinct specific inhibitors of PI3-K, wortmannin and LY294002, prevent VSMC migration induced by uPA. No migratory effect of uPA was observed in VSMC expressing the dominant negative form of Tyk2. Our results underscore the versatile function of Tyk2 in uPA-related intracellular signaling and indicate that PI3-K plays a selective role in the regulation of VSMC migration. Janus kinase signal transducers and activators of transcription urokinase-type plasminogen activator uPA receptor vascular smooth muscle cells phosphatidylinositol 3-kinase Src homology 2 polymerase chain reaction phosphate-buffered saline polyacrylamide gel electrophoresis A major pathway for signal generation by cytokines, growth factors, and polypeptide hormones involves activation of the Janus tyrosine kinase family (Jak kinases)1 and tyrosine phosphorylation of signal transducers and activators of transcription (Stat) proteins (1Ziemiecki A. Harpur A.G. Wilks A.F. Trends Cell Biol. 1994; 4: 207-212Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 2Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4912) Google Scholar). Jak activation results in tyrosine phosphorylation of several Stat proteins that form homo- and heterodimers and translocate to the nucleus to regulate gene transcription by binding to specific promoter sequences of stimulated genes (3Ihle J. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1258) Google Scholar). Which members of the Jak and Stat families are activated varies greatly among different agonists and in different cell systems (4Schindler C. Exp. Cell Res. 1999; 253: 7-14Crossref PubMed Scopus (114) Google Scholar, 5Ward A.C. Touw I. Yoshimura A. Blood. 2000; 95: 19-29Crossref PubMed Google Scholar). We have recently demonstrated that the urokinase receptor (uPAR) utilizes the Jak/Stat pathway for intracellular transmission in human vascular smooth muscle cells (VSMC) and endothelial cells (6Dumler I. Weis A. Mayboroda O.A. Maasch C. Jerke U. Haller H. Gulba D.C. J. Biol. Chem. 1998; 273: 315-321Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 7Dumler I. Kopmann A. Weis A. Mayboroda O.A. Wagner K. Gulba D.C. Haller H. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 290-297Crossref PubMed Scopus (55) Google Scholar). uPA/uPAR is a multifunctional system involved in wound healing, tissue remodeling, immune response, and cancer by affecting cell migration, adhesion, and proliferation (8Blasi F. Acta Pathol. Microbiol. Scand. 1999; 107: 96-101Crossref Scopus (84) Google Scholar, 9Chapman H.A. Curr. Opin. Cell Biol. 1997; 9: 714-724Crossref PubMed Scopus (421) Google Scholar). Some of these functions require uPAR-dependent signal transduction, which remains imperfectly defined. uPAR is associated with two Janus kinases, Jak1 and Tyk2, which become activated in response to uPA and subsequently induce formation and activation of Stat1, Stat2, and Stat4 complexes (6Dumler I. Weis A. Mayboroda O.A. Maasch C. Jerke U. Haller H. Gulba D.C. J. Biol. Chem. 1998; 273: 315-321Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 7Dumler I. Kopmann A. Weis A. Mayboroda O.A. Wagner K. Gulba D.C. Haller H. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 290-297Crossref PubMed Scopus (55) Google Scholar, 10Dumler I. Kopmann A. Wagner K. Mayboroda O.A. Jerke U. Dietz R. Haller H. Gulba D.C. J. Biol. Chem. 1999; 274: 24059-24065Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Both kinases co-localized with the uPAR to the leading edge of migrating VSMC (6Dumler I. Weis A. Mayboroda O.A. Maasch C. Jerke U. Haller H. Gulba D.C. J. Biol. Chem. 1998; 273: 315-321Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), implying a possible contribution of the Jaks to cell migration, most likely via the interactions with other kinases or signaling molecules. Several reports have shown that Jaks and Stats interfere with multiple signaling cascades, such as Ras/mitogen-activated protein kinase pathway, phosphatidylinositol 3-kinase (PI3-K), Pyk2, and Src kinases. These cascades couple Jak/Stat to pathways with different downstream signaling functions (5Ward A.C. Touw I. Yoshimura A. Blood. 2000; 95: 19-29Crossref PubMed Google Scholar, 11Chatterjee-Kishore M. Van den Aker F. Stark G.R. Trends Cell Biol. 2000; 10: 106-110Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). We reasoned that PI3-K is a candidate for mediating uPA-induced VSMC migration. PI3-K phosphorylates the D3 position of phosphatidylinositol, and the phosphorylated lipid products of this enzymatic reaction may act on multiple downstream effectors (12Kapeller R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar, 13Corvera S. Czech M.P. Trends Cell Biol. 1998; 8: 442-446Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). PI3-K is composed of two subunits, a regulatory p85 subunit and a 110-kDa catalytic subunit. The regulatory p85 subunit contains two Src homology 2 (SH2) domains, which bind to specific phosphotyrosine-containing motifs and have been implicated in mediating protein-protein interactions (14Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (825) Google Scholar). The ability of p85 SH2 domains to associate with other proteins links PI3-K to distinct signaling cascades required for control of cell growth and proliferation, adhesion and motility, differentiation, and survival (15Vanhaesebroeck B. Waterfield M.D. Exp. Cell Res. 1999; 253: 239-254Crossref PubMed Scopus (751) Google Scholar). Moreover, recent reports underscore the importance of PI3-K in cell migration (16Vanhaesebroeck B. Jones G.E. Allen W.E. Zicha D. Hooshmand-Rad R. Sawyer C. Wells C. Waterfield M.D. Ridley A.J. Nat. Cell Biol. 1999; 1: 69-71Crossref PubMed Scopus (198) Google Scholar, 17Reiske H.R. Kao S.C. Cary L.A. Guan J.L. Lai J.F. Chen H.C. J. Biol. Chem. 1999; 274: 12361-12366Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 18Adam L. Vadlamudi R. Kondapaka S.B. Chernoff J. Mendelson J. Kumar R. J. Biol. Chem. 1998; 273: 28238-28246Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). In this study, we investigated the extent to which PI3-K and Janus kinases are involved in the uPA-induced cell migration. Our findings demonstrate that in human VSMC, Tyk2, besides activation of Stat proteins in response to uPA, is the main uPA-dependent pathway of PI3-K activation. Tyk2 interacts with PI3-K via the SH2 domains of p85 catalytic subunit that, in turn, leads to p85 tyrosine phosphorylation and to PI3-K activation. The association of both kinases is critical to provide VSMC cytoskeletal reorganization in response to uPA required for cell migration. Chemicals of high quality commercial grade were purchased from Sigma, Amersham Pharmacia Biotech, Merck (Darmstadt, Germany), or Serva (Heidelberg, Germany). Radiochemicals and chemiluminescent signal enhancers were obtained from PerkinElmer Life Sciences. Vectashield mounting medium was purchased from Vector Laboratories, Inc. (Burlingame, CA). GST-(PI3-K)-p85-N-SH2 domain (amino acids 333–428) and GST-(PI3-K)-p85-C-SH2 domain (amino acids 624–718) were obtained as an agarose conjugate from Upstate Biotechnology, Inc. (Lake Placid, NY). Wortmannin and LY294002 were from Sigma; uPA and ATF were from Loxo (Dossenheim, Germany). Restriction endonucleases HindIII, XbaI,PacI, EheI, and T4 DNA ligase were from New England BioLabs (Beverly, CA); the Expand High Fidelity PCR system was from Roche Diagnostics GmbH (Mannheim, Germany); plasmid pCRI2.1TOPO was purchased from Invitrogen (Groningen, The Netherlands); and plasmids pTGBKCMV and pAD1 were from HepaVec (Berlin, Germany). Mono- and polyclonal anti-phosphotyrosine antibodies were from Affinity Research Products Ltd. (Exeter, UK) and Pierce; mono- and polyclonal anti-p85 PI3-K antibodies were from Transduction Laboratories (Lexington, KY) and Upstate Biotechnology, Inc. Mono- and polyclonal anti-Jak-kinase antibodies were purchased from Transduction Laboratories and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Peroxidase-conjugated affinipure goat anti-mouse and goat anti-rabbit IgG and Cy3-conjugated anti-rabbit and anti-mouse IgG were purchased from Dianova (Hamburg, Germany). Alexa 488-conjugated phalloidin was from Molecular Probes, Inc. (Eugene, OR). Human VSMC from coronary artery were obtained from Clonetics (San Diego, CA). The cells were grown in SmGM2 medium (Clonetics) supplemented with 5% fetal bovine serum and were used between passages 3 and 6. For uPA stimulation experiments, the cells were cultured for 24 h in serum-free medium and were then treated with uPA as described (6Dumler I. Weis A. Mayboroda O.A. Maasch C. Jerke U. Haller H. Gulba D.C. J. Biol. Chem. 1998; 273: 315-321Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Human transformed kidney epithelial cells HEK 293 were obtained from Microbix Biosystems (Toronto, Canada). Total RNA from human VSMC was extracted according to the acid phenol extraction protocol (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). Full-length wild type Tyk2 cDNA was performed by PCR on two pairs oligonucleotides (sense, 5′-GATGCCCGGGTCTGTGCTGAATG; antisense, 5′-CACGGCCAAGAAAGAAAAATAAGT) according to the high fidelity PCR protocol (Roche Diagnostics). The product of the reverse transcription reaction with tRNA was used as a template for PCR, and the PCR product was cloned into pCR2.1TOPO plasmid. The structure of Tyk2 was confirmed by sequencing. The recombinant adenovirus DNA for Tyk2 expression was generated by homologous recombination inEscherichia coli BJ5183 essentially as described previously (20Chartier C. Degryse E. Gantzer M. Dieterle A. Pavirani A. Mehtali M. J. Virol. 1996; 70: 4805-4810Crossref PubMed Google Scholar). Briefly, an HindIII–XbaI fragment of the plasmid pCRIITyk2 encoding wild type human Tyk2 was ligated intoXbaI–HindIII-digested pBKTGCMV. The resulting construct was cleaved with restriction endonucleases PacI and EheI to linearize it and to transform together with the linear adenoviral vector pAD1 in E. coli strain BJ5183. The recombinant adenovirus construct was cleaved with PacI and transfected into the packaging cell line HEK 293 by calcium phosphate precipitation (21Chen C. Okayama H. Mol. Cell Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar). The concentration of the recombinant adenovirus was assessed based on the absorbance at 260 nm and on limiting dilution plaque assay (22Becker T.C. Noel R.J. Coats W.S. Gomez-Foix A.M. Alam T. Gerard R.D. Newgard C.B. Methods Cell Biol. 1994; 43: 161-189Crossref PubMed Scopus (561) Google Scholar). An expression construct that encodes Tyk2 that is mutated in its active center at residue 3079 (Lys into Glu) was generated by site-directed mutagenesis using the QuickChange protocol from Stratagene (La Jolla, CA), and the corresponding recombinant adenovirus was prepared as indicated above. VSMC were grown to 50% confluency and infected for 1 h with recombinant AdTyk2 adenovirus stock at a multiplicity of infection of 500 plaque-forming units/cell. The efficiency of infection was assessed by immunological staining with anti-Tyk2 antibody. Cells were serum-starved overnight 1 day after infection and used for experiments on the second day after infection. Total RNA was prepared from VMSC at the indicated times after Ad5Tyk2 infection after 6 h of serum starvation using standard protocol with Trizol reagent (Life Technologies, Inc.). The RNA was electrophoresed through an agarose-formaldehyde gel, transferred to a gene-screen plus (DuPont, Bad Homburg, Germany), and hybridized with nick-translatedBamHI fragment from Tyk2 cDNA as described (23Dumler I. Petri T. Schleuning W.D. FEBS Lett. 1994; 343: 103-106Crossref PubMed Scopus (66) Google Scholar). An actin probe was used to confirm equal RNA loading on the gel. Subconfluent and serum-starved VSMC were treated with 1 nm uPA for 5–180 min at 37 °C, lysed, and precleared as described previously (6Dumler I. Weis A. Mayboroda O.A. Maasch C. Jerke U. Haller H. Gulba D.C. J. Biol. Chem. 1998; 273: 315-321Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). In some experiments, cells were pretreated with 100 nmwortmannin for 20 min at 37 °C. For immunoprecipitation, precleared cell lysates containing 800–1000 μg of protein were incubated overnight at 4 °C with 4–5 μg of antibody coupled to protein A-agarose. Precipitates were washed in PBS-Tween buffer and were used for PAGE and Western blotting. The blots were developed with the indicated antibodies, and the immune complexes were visualized by an enhanced chemiluminescence detection system. Membrane stripping was performed using 200 μm β-mercaptoethanol, 62.5 mm Tris-HCl, pH 6.8, and 2% SDS for 45 min at 50 °C. For the in vitro phosphorylation assay, 400–800 μg of protein of precleared cell lysates were incubated with 4–5 μg of the indicated antibody coupled to protein A-agarose for 2 h. Precipitates were washed four times in lysis buffer and twice in kinase buffer (20 mm HEPES, pH 7.4, 10 mm MgCl2, 5 mm MnCl2, 1 mm dithiothreitol, 300 μm sodium orthovanadate, 10 μg/ml leupeptin, 1 mmphenylmethylsulfonyl fluoride). The kinase assay was performed in 40 μl of kinase buffer containing 3–5 μCi of [γ-32P]ATP (3000 Ci/mmol) for 10 min at 30 °C. Precipitates were washed once with lysis buffer and twice in PBS-Tween buffer. The reaction product was eluted in 150 μl of lysis buffer containing 0.5% SDS and 5 mm sodium orthovanadate and then subjected to a second round of immunoprecipitation with 1 μg of the indicated antibody coupled to protein A-agarose overnight at 4 °C. Phosphorylated proteins were analyzed by SDS-PAGE and autoradiography. In some experiments, gels containing phosphorylated proteins were soaked in 1 n KOH at 55 °C for 2 h to hydrolyze phosphate on serine and threonine. Precleared cell lysates containing 600–800 μg of protein were incubated for 2 h at 4 °C with 4 μg of anti-PI3-K antibody against the p85 regulatory subunit and then precipitated on protein A-agarose. Precipitates were washed once with lysis buffer containing 300 μm sodium orthovanadate and 1 mm phenylmethylsulfonyl fluoride and then four times with 10 mm HEPES, 100 mm NaCl, pH 7.4, and then subjected to the in vitro kinase reaction in a final volume of 50 μl of reaction mixture containing 1 mg/mll-α-phosphatidylinositol, 3 mmMgCl2, 15 mm ATP, and 3–5 μCi of [γ-32P]ATP (3000 Ci/mmol) for 10 min at 30 °C. Phase separation of lipids was performed in two steps byn-hexane/isopropyl alcohol (26:14) and 2 nKCl/HCl (8:0.25). Phosphorylated lipids were separated by thin layer chromatography on aluminum sheets silica gel 60W (Merck, Germany) in chloroform/methanol/H2O/25% ammonium (45:35:7.5:2.8; v/v/v/v) and visualized by autoradiography. GST fusion proteins containing the single C- or N-terminal p85 SH2 domain (GST-p85-C-SH2 and GST-p85-N-SH2) bound to glutathione-agarose beads were used for affinity precipitation. Precleared cell lysates containing 400–800 μg of protein were incubated with 3–4 μg of SH2 domain conjugates for 2 h at 4 °C. Precipitates were washed three times with PBS-Tween buffer. Precipitated proteins were eluted with 2-fold Laemmli sample buffer containing 20 mm dithiothreitol and 10 mm glutathione and were used for PAGE and Western blotting. Chemotaxis assay was performed using modified Boyden chambers with polyvinylpyrrolidone-free polycarbonate filter membranes, 8-μm pore size, as described (24Degryse B. Resnati M. Rabbani S.A. Villa A. Fazioli F. Blasi F. Blood. 1999; 94: 649-662Crossref PubMed Google Scholar). 25,000–30,000 cells in serum-free SmGM2 medium were added to the upper well of the Boyden chamber. uPA was diluted in serum-free SmGM2 and added to the lower well of the Boyden chamber, and migration was allowed for 4 h. When chemotaxis was performed in the presence of the PI 3-K inhibitors wortmannin or LY294002, these substances were added to the upper well. All experiments were performed in triplicates. Cell migration was quantified by densitometry, and cell migration in the absence of chemoattractant was taken as 100%. Wounding experiments using confluent VSMC monolayer were performed as described previously (6Dumler I. Weis A. Mayboroda O.A. Maasch C. Jerke U. Haller H. Gulba D.C. J. Biol. Chem. 1998; 273: 315-321Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Before wounding, cells were cultured in serum-free SmGM2 medium for 6 h. In migration experiments using PI3-K inhibitors, cells were pretreated with wortmannin or LY294002 in serum-free medium for 30 min before wounding, and then inhibitors were added again to the serum-free medium after wounding together with or without uPA. Cell migration was monitored by time lapse imaging using an endoscope (telecam PAL 20210036, Storz, Germany) attached to an Axioplan microscope (Zeiss) and acquisition and analysis software (Avid Videoshop, Avid Desktop Software Inc., Microsoft Excel). The wounds were viewed inside an environmental chamber under constant temperature (37 °C) and humidified in 5% CO2 and air (CTI Controller 3700; Zeiss). Microscopic recordings were started immediately after wounding, and then further images were taken every 30 min for 9 h following wounding. Results are the mean number of migrated cells ± S.D. at indicated time points or -fold stimulation of at least five separate experiments. Cells were seeded and cultured on glass coverslips, and wounding was performed as indicated above. Cells were allowed to migrate for 8 h at 37 °C and then treated for different time periods with appropriate inhibitors or stimulators. After incubation, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, washed three times with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 3 min at room temperature. Then cells were stained with Alexa 488-conjugated phalloidin for 20 min at room temperature. Coverslips were mounted with Vectashield mounting medium. Immunofluorescent staining for Tyk2 was performed as described previously (6Dumler I. Weis A. Mayboroda O.A. Maasch C. Jerke U. Haller H. Gulba D.C. J. Biol. Chem. 1998; 273: 315-321Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Color images were captured using a SensiCam 12-bit CCD camera (PCO Computer Optics, Kelheim, Germany) and acquisition and analysis software (AxioVision and KS 300; Carl Zeiss, Vision, Munich, Germany) running on a Power PC 586/200MMX-64MB (Inteq, Berlin, Germany). Images for Alexa 488 and Cy3 staining were captured digitally and were imported as TIF files into Illustrator for analysis and printing. To address the potential role of PI3-K in uPA-mediated signaling, we first assayed PI3-K activity directly by measuring the levels of the product, phosphatidylinositol phosphate, in serum-starved VSMC stimulated with uPA at different time points. As shown in Fig. 1 A, uPA induced a significant increase (up to 3-fold) in PI3-K activity in a time-dependent manner with a sustained PI3-K activation observed after 3 h. The PI3-K-specific inhibitor wortmannin, which binds covalently to the catalytic p110 subunit of PI3-K and inhibits PI3-K irreversibly at nanomolar concentrations (12Kapeller R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar) completely blocked the uPA-induced stimulation of enzyme activity (Fig. 1 B). To assess a potential contribution of uPA's proteolytic activity to the mechanisms of PI3-K activation, the amino-terminal fragment of uPA, ATF, was used for cell stimulation. ATF provided the same effect (Fig. 1 C), confirming the involvement of proteolytically inactive uPA in signaling events. We also examined the requirement for uPAR upon PI3-K activation. For this purpose, we used soluble recombinant uPAR, which is a known competitor for the uPA- and ATF-uPAR binding. The data presented in Fig. 1 C clearly demonstrate that the pretreatment of VSMC with soluble recombinant uPAR significantly decreased the uPA/ATF-induced PI3-K stimulation (shown for ATF).Figure 1uPA induces PI3-K activation and tyrosine phosphorylation of p85 in VSMC. A, VSMC were treated with 1 nm uPA for the indicated times at 37 °C, and the PI3-K assay was performed with subsequent thin layer chromatography and autoradiography visualizing phosphatidylinositol 1,4,5-trisphosphate (PtdIns(3)P) products. B, cells were untreated or treated with uPA for 30 min and/or with wortmannin, as indicated. C, VSMC were stimulated with 1 nm ATF for 45 min in the presence or absence of 20 nm soluble recombinant uPAR. Recombinant soluble uPAR was expressed and purified as described previously (23Dumler I. Petri T. Schleuning W.D. FEBS Lett. 1994; 343: 103-106Crossref PubMed Scopus (66) Google Scholar). Quantification of the results by densitometry is shown below eachpanel. D, phosphorylation of p85 was assayed in control VSMC and after cell activation with 1 nm uPA byin vitro kinase assay combined with two rounds of immunoprecipitation (IP and re-IP) with anti-p85 polyclonal antibody (Ab), as described under “Experimental Procedures.” Phosphorylated proteins were subjected to SDS-PAGE and autoradiography before (upper panel) and after (middle panel) KOH treatment. The equal loading of precipitated proteins on the gel was confirmed by immunoblotting (lower panel). WB, Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next examined PI3-K p85 subunit phosphorylation after uPA stimulation using an in vitro kinase assay combined with two rounds of immunoprecipitation with anti-p85 antibody (Fig. 1 D, upper panel). The results demonstrate that uPA markedly increased phosphorylation of the p85 protein. The band was resistant to alkaline hydrolysis, indicating tyrosine phosphorylation (Fig. 1 D, middle panel). The equal loading of precipitated material on the gel was confirmed by immunoblotting (Fig. 1 D, lower panel). To examine the potential link between PI3-K and uPA-mediated Jak/Stat signaling, we first performed immunoprecipitation with anti-Jak antibodies and looked for the p85 subunit of PI3-K in the immunoprecipitates. Proteins that were co-precipitated with Jaks were subjected to in vitro kinase assay as above, and the phosphorylated proteins were reimmunoprecipitated with anti-p85 antibody. As a control for p85 recovery, both rounds of immunoprecipitation were performed with anti-p85 antibody. As shown in Fig. 2 A, upper panel, one band corresponding to p85 was detected in Tyk2, but not in Jak1, Jak2, or Jak3 immunoprecipitates. The identity of the immunoprecipitated p85 band was confirmed by immunoblotting with anti-p85 antibody (Fig. 2 A, lower panel). To verify that antibodies to Jak1, Jak2, and Jak3 were behaving appropriately under our experimental conditions, the additional positive controls were performed, confirming that all three kinases might be precipitated from the VSMC lysates (Fig. 2 B). These data demonstrate that p85 can directly and constitutively associate with Tyk2 in VSMC. Moreover, Tyk2 immunoprecipitates possessed PI3-K activity, which was enhanced by uPA stimulation (Fig. 2 C). To further characterize this interaction, we overexpressed the wild type Tyk2 in VSMC using adenoviral cell infection. The overexpression enabled us to obtain cell lysates enriched in Tyk2, which is otherwise expressed at a low level in native VSMC. Immunofluorescent staining of control uninfected cells and Ad5Tyk2-VSMC with anti-Tyk2 antibody confirmed the efficiency of cell infection with the recombinant adenovirus under these conditions (Fig. 3 A). Northern and Western blot analysis were further used to confirm the expression of Tyk2 mRNA and protein in VSMC infected with adenovirus and cultured for different time periods. As shown in Fig. 3 B, VSMC infection with Ad5Tyk2 resulted in a dramatic increase in Tyk2 mRNA and protein levels relative to uninfected cells. The Tyk2 protein expression reached a maximum 2 days after infection and decreased within the next 3 days. In further experiments, we therefore used VSMC cultured for 2 days after infection with Ad5Tyk2. To investigate the interaction mechanisms between Tyk2 and p85,in vitro binding experiments were performed. We used two GST fusion proteins containing the single C- and N-terminal Src homology 2 (SH2) domains of the human p85 subunit of PI3-K (GST-p85-C-SH2 and GST-p85-N-SH2) immobilized on the glutathione-agarose beads. The beads were incubated with the lysates of unstimulated and uPA-stimulated Ad5Tyk2-VSMC, and the precipitated proteins were analyzed by immunoblotting with anti-Tyk2 antibody. As shown in Fig. 3 C, one major band corresponding to Tyk2 was detected in both GST-p85-C-SH2 and GST-p85-N-SH2 precipitates from uPA-stimulated cells. Tyk2 bands identified in unstimulated cells were significantly weaker. These results strongly suggest that Tyk2 directly associates with PI3-K through the binding to both C- and N-terminal SH2 domains of p85 and that this association is uPA-inducible. Recent reports imply that PI3-K is involved in cell migration control (16Vanhaesebroeck B. Jones G.E. Allen W.E. Zicha D. Hooshmand-Rad R. Sawyer C. Wells C. Waterfield M.D. Ridley A.J. Nat. Cell Biol. 1999; 1: 69-71Crossref PubMed Scopus (198) Google Scholar, 17Reiske H.R. Kao S.C. Cary L.A. Guan J.L. Lai J.F. Chen H.C. J. Biol. Chem. 1999; 274: 12361-12366Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 18Adam L. Vadlamudi R. Kondapaka S.B. Chernoff J. Mendelson J. Kumar R. J. Biol. Chem. 1998; 273: 28238-28246Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Although uPA is known to regulate cell migration (25Blasi F. Immunol. Today. 1997; 18: 415-417Abstract Full Text PDF PubMed Scopus (240) Google Scholar), the underlying molecular mechanisms are unclear. Therefore, we next examined the possible impact of PI3-K on VSMC migration in response to uPA. For this purpose, two main approaches were used, namely directional VSMC migration in Boyden chambers and cell movement in a wounded VSMC monolayer monitored by time lapse imaging (see “Experimental Procedures” for details). Fig. 4 A displays the data on directional migration of VSMC along the uPA gradient tested in a microchemotaxis Boyden chamber. After 4 h, cell migration in response to uPA was significantly enhanced about 3-fold, compared with migration in the presence of medium alone. To examine the requirement of PI3-K for uPA-promoted cell migration, VSMC were subjected to migration assays in the presence of two unrelated structurally distinct specific PI3-K inhibitors, wortmannin and LY294002. Cell treatment with both inhibitors decreased uPA-related cell migration w" @default.
• W1969732387 created "2016-06-24" @default.
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• W1969732387 date "2000-12-01" @default.
• W1969732387 modified "2023-09-30" @default.
• W1969732387 title "Urokinase Stimulates Human Vascular Smooth Muscle Cell Migration via a Phosphatidylinositol 3-Kinase-Tyk2 Interaction" @default.
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