Thrombospondin Stimulates Focal Adhesion Disassembly through Gi- and Phosphoinositide 3-Kinase-dependent ERK Activation
2002; Elsevier BV; Volume: 277; Issue: 23 Linguagem: Inglês
10.1074/jbc.m112091200
ISSN1083-351X
AutoresA. Wayne Orr, Manuel A. Pallero, Joanne E. Murphy-Ullrich,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoThe matricellular protein thrombospondin (TSP) stimulates stress fiber and focal adhesion disassembly through a sequence (hep I) in its heparin-binding domain. TSP/hep I signals focal adhesion disassembly by binding cell surface calreticulin (CRT) and activating phosphoinositide 3-kinase (PI3K). However, other components of this signaling pathway have not been identified. We now show that TSP induces focal adhesion disassembly through activation of pertussis toxin (PTX)-sensitive G proteins and ERK phosphorylation. PTX pretreatment inhibits TSP/hep I-mediated focal adhesion disassembly as well as PI3K activation. In addition, membrane-permeable Gαi2- and Gβγ-blocking peptides inhibit hep I-mediated focal adhesion disassembly. Hep I stimulates a transient increase in ERK activation, which is abrogated by both PTX and PI3K inhibitors. Inhibiting ERK activation with MEK inhibitors blocks hep I-mediated focal adhesion disassembly, indicating that ERK activation is required for cytoskeletal reorganization. G protein signals and ERK phosphorylation are induced by TSP binding to cell surface CRT, because CRT null mouse embryonic fibroblasts (MEF) fail to stimulate ERK phosphorylation in response to TSP/hep I treatment. These data show that Gi protein and ERK, in concert with PI3K, are stimulated by TSP·CRT interactions at the cell surface to induce de-adhesive changes in the cytoskeleton. The matricellular protein thrombospondin (TSP) stimulates stress fiber and focal adhesion disassembly through a sequence (hep I) in its heparin-binding domain. TSP/hep I signals focal adhesion disassembly by binding cell surface calreticulin (CRT) and activating phosphoinositide 3-kinase (PI3K). However, other components of this signaling pathway have not been identified. We now show that TSP induces focal adhesion disassembly through activation of pertussis toxin (PTX)-sensitive G proteins and ERK phosphorylation. PTX pretreatment inhibits TSP/hep I-mediated focal adhesion disassembly as well as PI3K activation. In addition, membrane-permeable Gαi2- and Gβγ-blocking peptides inhibit hep I-mediated focal adhesion disassembly. Hep I stimulates a transient increase in ERK activation, which is abrogated by both PTX and PI3K inhibitors. Inhibiting ERK activation with MEK inhibitors blocks hep I-mediated focal adhesion disassembly, indicating that ERK activation is required for cytoskeletal reorganization. G protein signals and ERK phosphorylation are induced by TSP binding to cell surface CRT, because CRT null mouse embryonic fibroblasts (MEF) fail to stimulate ERK phosphorylation in response to TSP/hep I treatment. These data show that Gi protein and ERK, in concert with PI3K, are stimulated by TSP·CRT interactions at the cell surface to induce de-adhesive changes in the cytoskeleton. Cell adhesion is a key regulator of cellular physiology and pathophysiology, affecting the ability of a cell to proliferate, migrate, and even survive (1Huttenlocher A. Sandborg R.B. Horwitz A.F. Curr. Opin. Cell Biol. 1995; 7: 697-706Crossref PubMed Scopus (449) Google Scholar, 2Teti A. J. Am. Soc. Nephrol. 1992; 2: 582-587Google Scholar). Thus, modulation of cell adhesion potentially affects diverse aspects of cell behavior. One adhesion modulatory signal comes from a family of extracellular matrix proteins, termed matricellular proteins, which primarily stimulate anti-adhesive signals. The matricellular proteins, thrombospondin (TSP), 1The abbreviations used are: TSPthrombospondinADBassay dilution bufferERKextracellular signal-regulated kinasePTXpertussis toxinGPCRG protein-coupled receptorCRTcalreticulinMEFmouse embryonic fibroblastsBAEbovine aortic endothelialIAPintegrin-associated proteinPI3Kphosphoinositide 3-kinaseERendoplasmic reticulumJNKc-Jun N-terminal kinaseMAPKmitogen-activated protein kinaseEGFepidermal growth factorDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumMBPmyelin basic proteinMOPS4-morpholinepropanesulfonic acidPVDFpolyvinylidene difluorideHRPhorseradish peroxidaseSMCsmooth muscle cellPIP2phosphatidylinositol (4,5)-bisphosphatePIP3phosphatidylinositol (3,4,5)-trisphosphateIRMinterference reflection microscopyLRPlow density lipoprotein receptor-related protein1The abbreviations used are: TSPthrombospondinADBassay dilution bufferERKextracellular signal-regulated kinasePTXpertussis toxinGPCRG protein-coupled receptorCRTcalreticulinMEFmouse embryonic fibroblastsBAEbovine aortic endothelialIAPintegrin-associated proteinPI3Kphosphoinositide 3-kinaseERendoplasmic reticulumJNKc-Jun N-terminal kinaseMAPKmitogen-activated protein kinaseEGFepidermal growth factorDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumMBPmyelin basic proteinMOPS4-morpholinepropanesulfonic acidPVDFpolyvinylidene difluorideHRPhorseradish peroxidaseSMCsmooth muscle cellPIP2phosphatidylinositol (4,5)-bisphosphatePIP3phosphatidylinositol (3,4,5)-trisphosphateIRMinterference reflection microscopyLRPlow density lipoprotein receptor-related protein tenascin-C, and SPARC, can support varying degrees of cell adhesion (3Murphy-Ullrich J.E. J. Clin. Invest. 2001; 107: 785-790Crossref PubMed Scopus (401) Google Scholar, 4Sage E.H. Bornstein P. J. Biol. Chem. 1991; 266: 14831-14834Abstract Full Text PDF PubMed Google Scholar). However, a feature of these matricellular proteins is that they reduce cellular adhesiveness to a state of intermediate cell adhesion (3Murphy-Ullrich J.E. J. Clin. Invest. 2001; 107: 785-790Crossref PubMed Scopus (401) Google Scholar). This entails disassembly of focal adhesions, characterized by unbundling of actin stress fibers and selective depletion of certain focal adhesion proteins, including vinculin and α-actinin (3Murphy-Ullrich J.E. J. Clin. Invest. 2001; 107: 785-790Crossref PubMed Scopus (401) Google Scholar, 5Greenwood J.A. Murphy-Ullrich J.E. Microsc. Res. Tech. 1998; 43: 420-432Crossref PubMed Scopus (96) Google Scholar, 6Reuveni H. Geiger T. Geiger B. Levitzki A. J. Cell Biol. 2000; 151: 1179-1192Crossref PubMed Scopus (24) Google Scholar). However, in the intermediate adhesive state, integrins remain clustered and cell spreading is not appreciably altered (3Murphy-Ullrich J.E. J. Clin. Invest. 2001; 107: 785-790Crossref PubMed Scopus (401) Google Scholar). The functional significance of this adhesive state has yet to be defined, but it is reasonable to suggest that cells in this state have altered tensional forces. Current data suggest that focal adhesion disassembly can modulate the migratory capacity of the cell, affect cellular apoptosis, and induce alterations in gene expression (3Murphy-Ullrich J.E. J. Clin. Invest. 2001; 107: 785-790Crossref PubMed Scopus (401) Google Scholar). Despite the potential physiologic implications of cellular de-adhesion, very little is currently known about the cell surface receptors and intracellular signaling pathways that propagate focal adhesion disassembly in response to the matricellular proteins. thrombospondin assay dilution buffer extracellular signal-regulated kinase pertussis toxin G protein-coupled receptor calreticulin mouse embryonic fibroblasts bovine aortic endothelial integrin-associated protein phosphoinositide 3-kinase endoplasmic reticulum c-Jun N-terminal kinase mitogen-activated protein kinase epidermal growth factor Dulbecco's modified Eagle's medium fetal bovine serum myelin basic protein 4-morpholinepropanesulfonic acid polyvinylidene difluoride horseradish peroxidase smooth muscle cell phosphatidylinositol (4,5)-bisphosphate phosphatidylinositol (3,4,5)-trisphosphate interference reflection microscopy low density lipoprotein receptor-related protein thrombospondin assay dilution buffer extracellular signal-regulated kinase pertussis toxin G protein-coupled receptor calreticulin mouse embryonic fibroblasts bovine aortic endothelial integrin-associated protein phosphoinositide 3-kinase endoplasmic reticulum c-Jun N-terminal kinase mitogen-activated protein kinase epidermal growth factor Dulbecco's modified Eagle's medium fetal bovine serum myelin basic protein 4-morpholinepropanesulfonic acid polyvinylidene difluoride horseradish peroxidase smooth muscle cell phosphatidylinositol (4,5)-bisphosphate phosphatidylinositol (3,4,5)-trisphosphate interference reflection microscopy low density lipoprotein receptor-related protein Thrombospondin (TSP) is a large (180 kDa), homotrimeric, multidomain extracellular matrix glycoprotein. TSP binds several receptors on the cell surface, including heparan sulfate proteoglycans, calreticulin (CRT), CD36, integrin-associated protein (IAP), as well as the α3β1 and αvβ3 integrins, making TSP's role in physiology and pathophysiology complex (7Bornstein P. FASEB J. 1992; 6: 3290-3299Crossref PubMed Scopus (306) Google Scholar, 8Chen H. Herndon M.E. Lawler J. Matrix Biol. 2000; 19: 597-614Crossref PubMed Scopus (343) Google Scholar). The sequence in TSP responsible for inducing focal adhesion disassembly has been mapped to a 19-amino acid sequence in the N-terminal heparin-binding domain of TSP, termed the hep I sequence, because it lies within the first heparin-binding sequence of this domain (9Murphy-Ullrich J.E. Gurusiddappa S. Frazier W.A. Höök M. J. Biol. Chem. 1993; 268: 26784-26789Abstract Full Text PDF PubMed Google Scholar). Early work on the signaling of TSP-mediated focal adhesion disassembly illustrated that basal levels of PKG were required for this process (10Murphy-Ullrich J.E. Pallero M.A. Boerth N. Greenwood J.A. Lincoln T.M. Cornwell T.L. J. Cell Sci. 1996; 109: 2499-2508Crossref PubMed Google Scholar). More recent work showed that TSP and hep I induce focal adhesion disassembly by binding to cell surface CRT and activating PI3K (11Greenwood J.A. Pallero M.A. Theibert A.B. Murphy-Ullrich J.E. J. Biol. Chem. 1998; 273: 1755-1763Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 12Goicoechea S. Orr A.W. Pallero M.A. Eggleton P. Murphy-Ullrich J.E. J. Biol. Chem. 2000; 275: 36358-36368Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The hep I sequence of TSP binds to cell surface CRT, and this binding is necessary for hep I-mediated PI3K activation and focal adhesion disassembly (12Goicoechea S. Orr A.W. Pallero M.A. Eggleton P. Murphy-Ullrich J.E. J. Biol. Chem. 2000; 275: 36358-36368Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Although best known as an ER resident chaperone protein, CRT localizes to other cellular compartments, such as the cytosol and on the cell surface (13Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (671) Google Scholar, 14Gray A.J. Park P.W. Broekelmann T.J. Laurent G.J. Reeves J.T. Stenmark K.R. Mecham R.P. J. Biol. Chem. 1995; 270: 26602-26606Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15White T.K. Zhu Q. Tanzer M.L. J. Biol. Chem. 1995; 270: 15926-15929Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In addition to TSP, cell surface CRT propagates cellular responses to fibrinogen and glycosylated laminin, suggesting this surface form of CRT has a functional role as a cell surface receptor (14Gray A.J. Park P.W. Broekelmann T.J. Laurent G.J. Reeves J.T. Stenmark K.R. Mecham R.P. J. Biol. Chem. 1995; 270: 26602-26606Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15White T.K. Zhu Q. Tanzer M.L. J. Biol. Chem. 1995; 270: 15926-15929Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Although CRT is not a transmembrane protein, there is evidence that cell surface CRT engages intracellular signaling pathways. Cho et al. (16Cho J.-H. Homma K. Kanegasaki S. Natori S. Eur. J. Biochem. 1999; 266: 878-885Crossref PubMed Scopus (41) Google Scholar,17Cho J.-H. Homma K.J. Kanegasaki S. Natori S. Cell Stress Chaperones. 2001; 6: 148-152Crossref PubMed Scopus (22) Google Scholar) reported that cell surface CRT transmits the effects of an anti-microbial peptide on neutrophils and monocytes. The effects of this anti-microbial peptide are sensitive to pertussis toxin (PTX), a selective inhibitor of the Gi subclass of heterotrimeric G proteins, suggesting that CRT may act in conjunction with a G protein-coupled receptor to propagate intracellular signals. Pertussis toxin-sensitive G proteins are logical targets for the TSP-mediated focal adhesion disassembly response through CRT, because PTX-sensitive G proteins have previously been shown to stimulate focal adhesion and stress fiber disassembly in response to urokinase-type plasminogen activator and fibroblast-derived motility factor (18Degryse B. Resnati M. Rabbani S.A. Villa A. Fazioli F. Blasi F. Blood. 1999; 94: 649-662Crossref PubMed Google Scholar, 19Sugiura T. Shirasuna K. Hayashido Y. Sakai T. Matsuya T. Int. J. Cancer. 1996; 68: 774-781Crossref PubMed Scopus (20) Google Scholar). Two of the most common pathways stimulated downstream of PTX-sensitive G proteins are the PI3K and ERK signaling pathways (20Pierce K.L. Luttrell L.M. Lefkowitz R.J. Oncogene. 2001; 20: 1532-1539Crossref PubMed Scopus (365) Google Scholar, 21Sotsios Y. Ward S.G. Immunol. Rev. 2000; 177: 217-235Crossref PubMed Scopus (134) Google Scholar). Although PI3K is known to play a role in regulating cell adhesion by TSP, little is known about ERK's role in TSP signaling. Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 comprise the mitogen-activated protein kinase (MAPK) family of proteins (22Pearson G. Robinson F. Beers Gibson T. Xu B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3528) Google Scholar). ERK is activated by diverse stimuli and has been implicated in a wide range of cellular functions, including proliferation, migration, and survival (23Tibbles L.A. Woodgett J.R. Cell Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (553) Google Scholar). Although the role of adhesion in regulation of the ERK pathway has been widely studied, little is known concerning the effect of ERK signaling on adhesion itself. Signaling through the ERK pathway stimulates a decrease in integrin affinity, suggesting a negative feedback loop on cell adhesion (24Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). In addition, ERK has been shown to play a role in EGF-mediated focal adhesion disassembly, as well as focal adhesion disassembly induced by oncogenic Ras (25Xie H. Pallero M.A. Gupta K. Chang P. Ware M.F. Witke W. Kwiatkowski D.J. Lauffenburger D.A. Murphy-Ullrich J.E. Wells A. J. Cell Sci. 1998; 111: 615-624Crossref PubMed Google Scholar, 26Murphy-Ullrich J.E. Höök M. J. Cell Biol. 1989; 109: 1309-1319Crossref PubMed Scopus (198) Google Scholar). In these current studies, we characterize the role of PTX-sensitive G proteins and the ERK pathway in TSP-mediated focal adhesion disassembly. We show that TSP induces Gi-dependent PI3K and ERK activation, both of which are required for TSP-mediated focal adhesion disassembly. Furthermore, these data report, for the first time, the ability of cell surface CRT to mediate ERK activation. The following items were purchased: Dulbecco's modified Eagle's medium (DMEM, Cell-Gro, Mediatech, Herndon, VA), fetal bovine serum (FBS, HyClone Laboratories), 500 μg/ml trypsin, 2 mm EDTA (Invitrogen), prestained molecular weight markers (Bio-Rad), and a chemiluminescence PerkinElmer Life Sciences detection kit. Phosphatidylinositol (4,5)-bisphosphate (PIP2) was obtained from American Radiolabeled Chemicals (St. Louis, MO) and [32P]ATP was purchased from Amersham Biosciences, Inc. (Piscataway, NJ). Pertussis toxin was purchased from List Biological Laboratories Inc. (Campbell, CA). Wortmannin was purchased from Alexis Corp. (Switzerland), and LY294002 was purchased from BIOMOL (Plymouth Meeting, PA). U0126, PD98059, rabbit anti-phospho-ERK, and rabbit anti-ERK polyclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA). PY20, anti-phosphotyrosine antibodies, were purchased from BD Transduction Laboratories (Lexington, KY). Anti-MAPK 1/2 (ERK1/2-CT), dephosphorylated myelin basic protein (MBP), anti-phospho-MBP, protein kinase C inhibitor peptide, protein kinase A inhibitor peptide, and the p38 MAPK inhibitor SB202190 were purchased from Upstate Biotechnology (Lake Placid, NY). Compound R24571 was purchased from Sigma-Aldrich, Inc. (St. Louis, MO), and the JNK inhibitor SP600125 was purchased from Calbiochem (San Diego, CA). TSP was isolated from fresh human platelets purchased from the American Red Cross and purified as previously described using heparin affinity and gel filtration chromatography (6Reuveni H. Geiger T. Geiger B. Levitzki A. J. Cell Biol. 2000; 151: 1179-1192Crossref PubMed Scopus (24) Google Scholar). Recombinant tenascin fnIIIA-D was a gift of Dr. Harold Erickson, Duke University (27Aukhil I. Joshi P. Yan Y. Erickson H.P. J. Biol. Chem. 1993; 268: 2542-2553Abstract Full Text PDF PubMed Google Scholar). Hep I (ELTGAARKGSGRRLVKGPDC) and modified hep I (ELTGAARAGSGRRLVAGPDC) peptides were synthesized, purified, and analyzed by the University of Alabama at Birmingham Comprehensive Cancer Center/Peptide Synthesis and Analysis shared facility and by Anaspec, Inc. (San Jose, CA). The membrane-permeable sequence (AAVALLPAVLLALLAK), MPS-Gαi2(AAVALLPAVLLALLAKKNNLKDCGLF), MPS-Gαi3(AAVALLPAVLLALLAKKNNLKECGLY), and MPS-Phosducin-like protein C terminus (Phos) (AAVALLPAVLLALLAKVTDQLGEDFFAVDLEAFLQEFGLLPEKE) were synthesized, purified, and analyzed by Anaspec, Inc. (San Jose, CA). BAE cells were isolated and cultured in DMEM containing 4.5 g/liter glucose, 2 mm glutamine, and 20% fetal bovine serum (FBS) as described previously (9Murphy-Ullrich J.E. Gurusiddappa S. Frazier W.A. Höök M. J. Biol. Chem. 1993; 268: 26784-26789Abstract Full Text PDF PubMed Google Scholar). Mouse embryonic fibroblasts (K41 (wild-type), K42 (CRT-knockout), and K42 (CRT-rescued)) were a gift of Dr. Marek Michalak, University of Alberta, Edmonton, Alberta, Canada (28Mesaeli N. Nakamura K. Zvaritch E. Dickie P. Dziak E. Krause K.H. Opas M. MacLennan D.H. Michalak M. J. Cell Biol. 1999; 144: 857-868Crossref PubMed Scopus (424) Google Scholar). Growth conditions were the same as described for BAE cells. Focal adhesion assays were performed as previously described (6Reuveni H. Geiger T. Geiger B. Levitzki A. J. Cell Biol. 2000; 151: 1179-1192Crossref PubMed Scopus (24) Google Scholar). Briefly, BAE and MEF cells were grown overnight on glass coverslips in DMEM with 10% FBS. After overnight incubation, cells were ∼70% to 80% confluent. Cells were then washed once with serum-free DMEM and incubated in serum-free DMEM for 30 min. Cells were then treated with either DMEM, hep I (1 μm), or TSP (10 μg/ml = 78 nmmonomer), fixed with 3% glutaraldehyde, and examined using a Zeiss Axiovert 10 equipped for interference reflection microscopy. Cells were scored as either positive or negative for the presence of focal adhesions, with cells containing at least five focal adhesions considered positive. At least 300 cells were evaluated for each condition. To prepare whole cell lysates from BAE and MEF cells grown in six-well plates, medium was removed from cells and 100 μl of SDS sample buffer (62.5 mm Tris-HCl (pH 6.8), 2% w/v SDS, 10% glycerol, 50 mm dithiothreitol, 0.1% w/v bromphenol blue) was added to each well. Following lysis is SDS sample buffer, lysates were harvested with cell scrapers and collected in Eppendorf tubes. Lysates were sonicated for 15 s to shear DNA and lower viscosity. Lysates were then boiled, centrifuged, and frozen at −20 °C until gel electrophoresis was performed. Medium was removed, and 1 ml of lysis buffer (10 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EGTA, 1 mm EDTA, 2 mm Na3VO4, 1% Triton X-100, 0.5% Nonidet P-40, 1 μg/ml leupeptin, and 1 μg/ml aprotinin) was added. Cells were scrapped, collected in Eppendorf tubes, and pre-cleared by centrifugation. Supernatants were incubated with PY20 (10 μg/ml) antibodies for 2 h on ice. Protein A-Sepharose was added for 1 h at 4 °C with shaking. Immunoprecipitates were washed three times with lysis buffer and twice in kinase buffer (10 mmHEPES, pH 7.2, 20 mm β-glycerophosphate, 0.8 mm Na3VO4, and 30 mmNaCl). Lipids were prepared by adding 400 μl of kinase buffer with 3.5 mm dithiothreitol to a pre-dried equal mixture of PIP2 and phosphatidylserine, to achieve a final 1.5 μm concentration of each lipid. Kinase buffer was removed from immunoprecipitates, and 20 μl of lipids was added to each tube and incubated for exactly 10 min at 37 °C. Next, 20 μl of reaction buffer (kinase buffer containing 17.5 μm ATP, 25 μCi of [32P]ATP/sample, and 17.5 mmMgCl2) was added to each tube and incubated at 37 °C for 10 min. The reaction was stopped by adding 160 μl of a 1:1 methanol/chloroform solution. Lipids were extracted by adding 80 μl of HCl to each tube and centrifuging to separate the phases. The lower phase was removed and lipids were separated by TLC on Silica Gel 60 plates pre-coated with 1% potassium oxalate. The plates were developed in chloroform/acetone/methanol/acetic acid/water (40:15:13:12:7), exposed for autoradiography, and quantified using Scanalytic's One-Dscan version 1.31. BAE cells were grown to near confluence and treated with hep I (1 μm) for various time points. Cells were then harvested by addition of lysis buffer (with 25 mm NaF and 1 mm phenylmethylsulfonyl fluoride). Anti-MAPK 1/2 antibodies were preincubated with Protein A-Sepharose beads for 2 h at 4 °C and washed three times with lysis buffer prior to use. Cell lysates were incubated with the anti-MAPK conjugated beads for 2 h and washed three times with lysis buffer. Immunoprecipitates were then washed two times with assay dilution buffer (20 mm MOPS, pH 7.2, 25 mmβ-glycerophosphate, 5 mm EGTA, 1 mm sodium orthovanadate, 1 mm dithiothreitol). Buffer was removed, and immunoprecipitates were sequentially given 10 μl of inhibitor mixture (20 μm protein kinase C inhibitor peptide, 2 μm protein kinase A inhibitor peptide, 20 μm compound R24571 in ADB), 10 μl of substrate mixture (2 mg/ml dephosphorylated myelin basic protein (MBP) in ADB), and 10 μl of magnesium/ATP mixture (500 μm ATP and 75 mm MgCl2 in ADB). Tubes were vortexed, and reaction was allowed to proceed for 20 min at 30 °C with agitation. Reaction was stopped by addition of 8 μl of 6× sample buffer. Samples were analyzed for phosphorylated MBP by immunoblotting. Samples were separated by gel electrophoresis using 12% SDS-PAGE gels for ERK and 15% SDS-PAGE gels for MBP and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk and immunoblotted with either 1:1000 anti-ERK antibodies, 1:1000 anti-phospho-ERK antibodies, or 1:1000 anti-phospho-MBP antibodies overnight. HRP-conjugated goat anti-rabbit IgG was used at a 1:5000 dilution for 1 h. Bands were visualized by incubating antibody-labeled membranes with PerkinElmer Life Sciences chemiluminescence reagent and exposing for autoradiography. Bands were quantified using Scanalytic's One-Dscan version 1.31, and phospho-ERK levels were normalized to levels of total ERK protein. Statistical analysis was performed using unpaired Student's t test, and p < 0.05 or p < 0.01 (indicated) was considered significant. We previously characterized a role for cell surface CRT in TSP/hep I-mediated focal adhesion disassembly and PI3K activation, suggesting that CRT is acting as a receptor for the hep I sequence of TSP (12Goicoechea S. Orr A.W. Pallero M.A. Eggleton P. Murphy-Ullrich J.E. J. Biol. Chem. 2000; 275: 36358-36368Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). However, CRT is not a transmembrane protein and most likely associates with a transmembrane protein to generate an intracellular signal. Because cell surface CRT has been shown to signal through a G protein-dependent mechanism in neutrophils and monocytes (16Cho J.-H. Homma K. Kanegasaki S. Natori S. Eur. J. Biochem. 1999; 266: 878-885Crossref PubMed Scopus (41) Google Scholar, 17Cho J.-H. Homma K.J. Kanegasaki S. Natori S. Cell Stress Chaperones. 2001; 6: 148-152Crossref PubMed Scopus (22) Google Scholar), we sought to determine what role heterotrimeric G proteins might play in TSP-induced focal adhesion disassembly. Pertussis toxin catalyzes the ADP-ribosylation and inactivation of the Gi/o subclass of heterotrimeric G proteins (29McKenzie F.R. Kelly E.C. Unson C.G. Spiegel A.M. Milligan G. Biochem. J. 1988; 249: 653-659Crossref PubMed Scopus (39) Google Scholar). To determine if pertussis-toxin sensitive G proteins are involved in TSP/hep I-mediated focal adhesion disassembly, we observed the effect of pertussis toxin (25 ng/ml for 12 h) pretreatment on TSP and hep I-mediated focal adhesion disassembly. Pretreatment of BAE cells with pertussis toxin blocked the ability of both TSP and hep I to stimulate a decrease in the percentage of focal adhesion-positive cells as measured by IRM (Fig. 1A). Pertussis toxin did not affect the basal level of focal adhesion-positive cells. The active sequence (TNfnIIIA-D) of tenascin-C (27Aukhil I. Joshi P. Yan Y. Erickson H.P. J. Biol. Chem. 1993; 268: 2542-2553Abstract Full Text PDF PubMed Google Scholar), another matricellular protein, retained the ability to induce focal adhesion disassembly in the pertussis toxin-treated cells (Fig. 1B), suggesting the effect of pertussis toxin is specific for TSP. In addition, pertussis toxin inactivated by boiling did not inhibit TSP/hep I-mediated focal adhesion disassembly (data not shown). Cholera toxin, which activates the Gs subclass of heterotrimeric G proteins, had no affect on focal adhesion disassembly in response to hep I, suggesting that TSP signals focal adhesion disassembly specifically through the Gi/o subclass of heterotrimeric G proteins (data not shown). Since activation of PI3K by TSP/hep I was shown to be essential for focal adhesion disassembly (11Greenwood J.A. Pallero M.A. Theibert A.B. Murphy-Ullrich J.E. J. Biol. Chem. 1998; 273: 1755-1763Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), we also sought to determine whether pertussis toxin could inhibit the ability of TSP/hep I to activate PI3K. BAE cells were serum-deprived in the presence or absence of 25 ng/ml PTX for 12 h, stimulated with hep I, TSP, or a modified hep I peptide, and the resulting PI3K activity was assessed. Although pertussis toxin slightly raised the basal level of PI3K activity, pertussis toxin treatment significantly inhibited the ability of both TSP and hep I to stimulate PI3K activation (Fig. 2). A modified form of the hep I peptide, with the essential lysines at positions 24 and 32 converted to alanine residues, did not stimulate PI3K activation under either condition. Pertussis toxin does not generally affect PI3K activity, because insulin stimulated PI3K activation was not inhibited by pertussis toxin (data not shown). These data provide evidence for the involvement of heterotrimeric G proteins in TSP/hep I-mediated focal adhesion disassembly. BAE cells contain only two known pertussis toxin-sensitive G proteins, Gαi2 and Gαi3 (30Jo H. Sipos K., Go, Y.-M. Law R. Rong J. McDonald J. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). To further characterize the involvement of these proteins in TSP and hep I-mediated focal adhesion disassembly, we employed a membrane-permeable peptide approach to specifically block signaling through Gαi2, Gαi3, and their associated Gβγ subunits. The C-terminal 10 amino acids from the Gα subunits have previously been demonstrated to specifically block the G protein-receptor interaction (31Taylor J.M. Neubig R.R. Cell. Signal. 1994; 6: 841-849Crossref PubMed Scopus (40) Google Scholar). Thus, the C-terminal 10 amino acids from Gαi2 and Gαi3 were produced coupled to the membrane-permeable sequence from Kaposi fibroblast growth factor. This approach has proven successful in blocking G protein signaling, delivering the inhibitory peptides into every cell type tested to date (32Chang M. Zhang L. Tam J.P. Sanders-Bush E. J. Biol. Chem. 2000; 275: 7021-7029Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 33Chang, M. S. S., Tam, J. P., and Sanders-Bush, E. (2000)Science's STKE, www.stke.org/cgi/content/full/OC_sigtrans;2000/47/p11Google Scholar). In addition, a Gβγ-sequestering 28-amino acid peptide from the C terminus of phosducin-like protein, a known Gβγ signaling inhibitor, was also produced and coupled to the membrane-permeable sequence (32Chang M. Zhang L. Tam J.P. Sanders-Bush E. J. Biol. Chem. 2000; 275: 7021-7029Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 33Chang, M. S. S., Tam, J. P., and Sanders-Bush, E. (2000)Science's STKE, www.stke.org/cgi/content/full/OC_sigtrans;2000/47/p11Google Scholar). BAE cells were incubated for 1 h with the various peptides (1 μm), treated with DMEM or hep I for 30 min, and the percentage of focal adhesion-positive cells was assessed by interference reflection microscopy. Pretreatment with both the Gαi2 and the Gβγ inhibitory peptides was able to inhibit hep I-mediated focal adhesion disassembly (Fig. 3). Interestingly, the Gαi3inhibitory peptide was not able to block this effect, although its functional sequence differs from that of the Gαi2inhibitory peptide in only 2 of the 10 amino acids. The membrane-permeable sequence alone had no effect on hep I-induced focal adhesion disassembly, suggesting that the effect is specific for the G protein-inhibitory sequence. Pertussis toxin-sensitive G proteins have been shown to stimulate activation of the ERK signaling pathway in response to a variety of agonists (20Pierce K.L. Luttrell L.M. Lefkowitz R.J. Oncogene. 2001; 20: 1532-1539Crossref PubMed Scopus (3
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