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• W2039926575 abstract "β-Arrestins are pleiotropic molecules that mediate signal desensitization, G-protein-independent signaling, scaffolding of signaling molecules, and chemotaxis. Protease-activated receptor-2 (PAR-2), a Gαq/11-coupled receptor, which has been proposed as a therapeutic target for inflammation and cancer, requires the scaffolding function of β-arrestins for chemotaxis. We hypothesized that PAR-2 can trigger specific responses by differential activation of two pathways, one through classic Gαq/Ca2+ signaling and one through β-arrestins, and we proposed that the latter involves scaffolding of proteins involved in cell migration and actin assembly. Here we demonstrate the following. (a) PAR-2 promotes β-arrestin-dependent dephosphorylation and activation of the actin filament-severing protein (cofilin) independently of Gαq/Ca2+ signaling. (b) PAR-2-evoked cofilin dephosphorylation requires both the activity of a recently identified cofilin-specific phosphatase (chronophin) and inhibition of LIM kinase (LIMK) activity. (c) β-Arrestins can interact with cofilin, LIMK, and chronophin and colocalize with them in membrane protrusions, suggesting that β-arrestins may spatially regulate their activities. These findings identify cofilin as a novel target of β-arrestin-dependent scaffolding and suggest that many PAR-2-induced processes may be independent of Gαq/11 protein coupling. β-Arrestins are pleiotropic molecules that mediate signal desensitization, G-protein-independent signaling, scaffolding of signaling molecules, and chemotaxis. Protease-activated receptor-2 (PAR-2), a Gαq/11-coupled receptor, which has been proposed as a therapeutic target for inflammation and cancer, requires the scaffolding function of β-arrestins for chemotaxis. We hypothesized that PAR-2 can trigger specific responses by differential activation of two pathways, one through classic Gαq/Ca2+ signaling and one through β-arrestins, and we proposed that the latter involves scaffolding of proteins involved in cell migration and actin assembly. Here we demonstrate the following. (a) PAR-2 promotes β-arrestin-dependent dephosphorylation and activation of the actin filament-severing protein (cofilin) independently of Gαq/Ca2+ signaling. (b) PAR-2-evoked cofilin dephosphorylation requires both the activity of a recently identified cofilin-specific phosphatase (chronophin) and inhibition of LIM kinase (LIMK) activity. (c) β-Arrestins can interact with cofilin, LIMK, and chronophin and colocalize with them in membrane protrusions, suggesting that β-arrestins may spatially regulate their activities. These findings identify cofilin as a novel target of β-arrestin-dependent scaffolding and suggest that many PAR-2-induced processes may be independent of Gαq/11 protein coupling. β-Arrestins were originally identified as terminators of G-protein-coupled receptor signaling and later as signaling scaffolds that regulate activation, subcellular localization, and signal duration of mitogen-activated protein kinases (MAPKs). Over the past 5 years, siRNA 2The abbreviations used are: siRNA, small inhibitory RNA; CIN, chronophin; DKO, double knock-out; ERK, extracellular signal-regulated kinase; IF, immunofluorescence; MEF, mouse embryonic fibroblast; PAR-2, protease-activated receptor-2; WB, Western blot; IP, immunoprecipitation; GST, glutathione S-transferase; BAPTA-AM, 1,2-bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); PLC, phospholipase C; GP, G-protein; 2fAP, 2-furoyl-LIGRL-ornithine-NH2; GFP, green fluorescent protein; wt, wild type; DN, dominant negative. studies silencing individual β-arrestins have revealed both redundant and selective roles for either β-arrestin-1 or -2 in the regulation of additional signaling molecules such as phosphatidylinositol 3-kinase and RhoA (1Shenoy S.K. Lefkowitz R.J. Biochem. J. 2003; 375: 503-515Crossref PubMed Scopus (339) Google Scholar, 2Lefkowitz R.J. Shenoy S.K. 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Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (555) Google Scholar, 17Shenoy S.K. Drake M.T. Nelson C.D. Houtz D.A. Xiao K. Madabushi S. Reiter E. Premont R.T. Lichtarge O. Lefkowitz R.J. J. Biol. Chem. 2005; 271: 1261-1273Google Scholar), highlighting the importance of identifying the signaling cascades they regulate. Protease-activated receptor-2 (PAR-2), which requires β-arrestins for desensitization, localization of ERK1/2, and cell migration, was one of the first G-protein-coupled receptors shown to utilize β-arrestin-dependent scaffolding (9Ge L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 10Ge L. Shenoy S.K. Lefkowitz R.J. DeFea K.A. J. Biol. Chem. 2004; 279: 55419-55424Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). PAR-2 is reported to trigger a wide variety of cellular responses (e.g. proliferation, chemotaxis, ion transport, epithelial barrier function, and tumor cell metastasis) in a cell type-specific manner (10Ge L. Shenoy S.K. Lefkowitz R.J. DeFea K.A. J. Biol. Chem. 2004; 279: 55419-55424Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 18Bolton S.J. McNulty C.A. Thomas R.J. Hewitt C.R. Wardlaw A.J. J. Leukoc. Biol. 2003; 74: 60-68Crossref PubMed Scopus (69) Google Scholar, 19Cenac N. Garcia-Villar R. Ferrier L. Larauche M. Vergnolle N. Bunnett N.W. Coelho A.M. Fioramonti J. Bueno L. J. Immunol. 2003; 170: 4296-4300Crossref PubMed Scopus (131) Google Scholar, 20Chambers L.S. Black J.L. Poronnik P. Johnson P.R. Am. J. Physiol. 2001; 281: L1369-L1378Crossref PubMed Google Scholar, 21Cuffe J.E. Bertog M. Velazquez-Rocha S. Dery O. Bunnett N. Korbmacher C. J. Physiol. 2002; 539: 209-222Crossref PubMed Scopus (56) Google Scholar, 22Ferrell W.R. Lockhart J.C. Kelso E.B. 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Cancer Biol. 2001; 11: 119-128Crossref PubMed Scopus (117) Google Scholar, 30Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar) and β-arrestins are capable of spatially restricting activity of signaling molecules downstream of PAR-2 activation, we hypothesized that β-arrestins might contribute to the spatial and temporal control over actin assembly/disassembly through scaffolding of signaling molecules traditionally associated with chemotaxis. We focused on the actin filament-severing protein cofilin, the activity of which allows for rapid changes in cytoskeletal architecture, creation of new barbed ends for elongation, and recycling of ATP monomers (28Cramer L.P. Mitchison T.J. Theriot J.A. Curr. Opin. Cell Biol. 1994; 6: 82-86Crossref PubMed Scopus (108) Google Scholar, 30Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar, 31DesMarais V. Ghosh M. Eddy R. Condeelis J. J. Cell Sci. 2005; 118: 19-26Crossref PubMed Scopus (249) Google Scholar, 32Dawe H.R. Minamide L.S. Bamburg J.R. Cramer L.P. Curr. Biol. 2003; 13: 252-257Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 33Bamburg J.R. Wiggan O.P. Trends Cell Biol. 2002; 12: 598-605Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Cofilin activation is controlled by opposing actions of LIMKs (which inactivate it by phosphorylation on Ser3) and cofilin-specific phosphatases (chronophin (CIN) and slingshot) that activate it (33Bamburg J.R. Wiggan O.P. Trends Cell Biol. 2002; 12: 598-605Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 34Nagata-Ohashi K. Ohta Y. Goto K. Chiba S. Mori R. Nishita M. Ohashi K. Kousaka K. Iwamatsu A. Niwa R. Uemura T. Mizuno K. J. Cell Biol. 2004; 165: 465-471Crossref PubMed Scopus (160) Google Scholar, 35Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (849) Google Scholar, 36Gohla A. Birkenfeld J. Bokoch G.M. Nat. Cell Biol. 2005; 7: 21-29Crossref PubMed Scopus (257) Google Scholar) as well as by intracellular pH and phosphatidylinositol 4,5-bisphosphate levels (37Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (844) Google Scholar). In the studies described here, we investigated the following: 1) whether PAR-2 promotes cofilin dephosphorylation and activation through a β-arrestin-dependent mechanism, 2) whether β-arrestins can scaffold and direct subcellular localization of cofilin, 3) whether β-arrestin-dependent cofilin activation is independent of classic Gαq signaling, and 4) whether upstream cofilin regulators CIN and LIMK are regulated by β-arrestins in response to PAR-2 activation. All chemicals were from Sigma unless otherwise stated. Antibodies and final dilutions for Western blot (WB), immunoprecipitation (IP) and immunofluorescence (IF) were as follows: from Chemicon, rabbit anti-phospho (Ser3)-cofilin (1:100 for IF) and mouse anti-cofilin (4 μg/ml for IP); from BD Pharmingen, mouse monoclonal antibodies to cofilin (1:1000 for WB and 1:250 for IF) and β-arrestin-1 (1:1000 for WB); from Dr. Robert J. Lefkowitz (Duke University Medical Center), affinity-purified rabbit antibody to β-arrestin-1 + 2 (A1CT; 1:500 for WB and 1:200 for IF) or β-arrestin-2 (A2CT, which recognizes β-arrestin-1 weakly as well; 1:500 for WB); from Cell Signaling Technology Inc., rabbit phospho-LIMK1/2 (1:500 for WB) and rabbit anti-phospho (Ser3)-cofilin (1:1000 for WB); from Santa Cruz Biotechnology, goat anti-actin (1:2000 for WB), mouse monoclonal anti-LIMK C10 (2 μg/ml for IP), rabbit anti-Myc A14 (1.5 μg/ml for IP, 1:200 for IF, and 1:1000 for WB), anti-Myc 9E10 (1:1000 for WB), and rabbit anti-Gαq/11 (1:1000 for WB); from Molecular Probes, Alexa488, Alexa595, and Alexa680-conjugated secondary antibodies (1:1000 for IF and 1:45,000 for WB); from Rockland, IRDye800-conjugated secondary antibodies (1:45,000 for WB); and from Biomeda, rabbit-anti-FLAG (1:1000 for WB and 1.5 μg/ml for IP). Rabbit polyclonal antibody to CIN has been described previously (36Gohla A. Birkenfeld J. Bokoch G.M. Nat. Cell Biol. 2005; 7: 21-29Crossref PubMed Scopus (257) Google Scholar). Activating peptides AP (SLIGRL-NH2 for mouse tethered ligand) and 2fAP (2-furoyl-LIGRL-ornithine-NH2) were synthesized by Genemed Inc. (South San Francisco, CA). Fura-2/AM was from Molecular Probes. Muscle pyrene-actin was from Cytoskeleton Inc. Recombinant activated LIMK (amino acids 285-639) and recombinant GST-cofilin were from Upstate. Pharmacological inhibitors were used as follows: Ca2+ chelator BAPTA-AM (Sigma) was prepared in N,N-dimethylformamide and used at 30 μm final concentration, PLC inhibitor U72133 (Tocris) was solubilized in chloroform and reconstituted to 100 mm in Me2SO immediately before use at 1 μm final concentration, and GP antagonist 2A (GP2A) was solubilized at 10 mm in Me2SO and used at a final concentration of 10 μm. Cells were permeabilized in Me2SO before addition of U72133 and GP2A. MEFs from wild type (MEFwt) and β-arrestin-1/2-/- knock-out mice (MEFβarrDKO) were gifts from Dr. Robert Lefkowitz (Duke University Medical Center) and have been described previously (38Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar). MDA-MB-468 cells were purchased from ATCC and grown in Dulbecco's modified Eagle's medium and 10% fetal calf serum. FLAG- and GFP-tagged β-arrestin-1 and -2 were a gift from Dr. Robert Lefkowitz (Duke University Medical Center) and have been described previously (39Dery O. Thoma M.S. Wong H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 18524-18535Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 40Stalheim L. Ding Y. Gullapalli A. Paing M.M. Wolfe B.L. Morris D.R. Trejo J. Mol. Pharmacol. 2005; 67: 78-87Crossref PubMed Scopus (80) Google Scholar). Dominant negative CIN and Myc-tagged CIN constructs have been described previously (36Gohla A. Birkenfeld J. Bokoch G.M. Nat. Cell Biol. 2005; 7: 21-29Crossref PubMed Scopus (257) Google Scholar). Transient transfections were performed on 70-80% confluent cells using Lipofectamine (Invitrogen), and experiments were performed between 24 and 48 h of transfection. 5 × 105 cells/60-mm dish (grown for 24 h) were serum-starved for 2 h, treated with 100 nm 2fAP for 0-90 min at 37 °C, lysed in 0.25 ml of lysis buffer (phosphate-buffered saline, pH 7.6, 1% Triton X-100, 1 mm EGTA, 2 mm NaF, 100 μm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml each aprotinin, leupeptin, and benzamidine). 25 μg of cleared lysate protein was analyzed by SDS-PAGE (15% for cofilin, 10% for LIMK, and 12.5% for β-arrestin) followed by Western blotting. Blots were imaged using the LICOR Odyssey imaging system, and LICOR software was used to calculate integrated intensities of bands. Images of Western blots were assembled using Adobe Photoshop 5.0 and imported into Canvas. Some gels were spliced to eliminate blank lanes or lanes containing samples unrelated to the figure. Two sets of oligos were used to knock down β-arrestins. The first set were chemically synthesized double-stranded siRNAs (β-arrestin-1, 5′-AAAGCCUUCUGCGCGGAGAAU-3′; β-arrestin-2, 5′-AAGGACCGCAAAGUGUUUGUG-3′; β-arrestin-1 and -2, 5′-ACCUGCGCCUUCCGCUAUG-3′; control (non-targeting sequence), 5′-AAUUCUCCGAACGUGUCACGU-3′) with 19-nucleotide duplex RNA and 2-nucleotide 3′ dT overhangs purchased from City of Hope Beckman Research Center (Duarte, CA) and Sigma Genosys in deprotected and desalted form. Specificity of these siRNA sequences for each β-arrestin has been validated previously (10Ge L. Shenoy S.K. Lefkowitz R.J. DeFea K.A. J. Biol. Chem. 2004; 279: 55419-55424Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 16Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (555) Google Scholar, 17Shenoy S.K. Drake M.T. Nelson C.D. Houtz D.A. Xiao K. Madabushi S. Reiter E. Premont R.T. Lichtarge O. Lefkowitz R.J. J. Biol. Chem. 2005; 271: 1261-1273Google Scholar, 40Stalheim L. Ding Y. Gullapalli A. Paing M.M. Wolfe B.L. Morris D.R. Trejo J. Mol. Pharmacol. 2005; 67: 78-87Crossref PubMed Scopus (80) Google Scholar, 41Ahn S. Nelson C.D. Garrison T.R. Miller W.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1740-1744Crossref PubMed Scopus (192) Google Scholar, 42Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 43Usui I. Imamura T. Huang J. Satoh H. Shenoy S.K. Lefkowitz R.J. Hupfeld C.J. Olefsky J.M. Mol. Cell. Biol. 2004; 24: 8929-8937Crossref PubMed Scopus (54) Google Scholar). A second set of siRNA oligos for each β-arrestin-1 and -2 and siRNA to human Gαq were obtained from Santa Cruz Biotechnology, and those sequences are proprietary. Each knockdown experiment described herein was confirmed by quantitative Western blot analysis (LICOR Odyssey infrared imaging system) using an internal loading control (ERK1/2, tubulin, or actin). Specificity of β-arrestin siRNA was further confirmed using antibodies that recognize both β-arrestin-1 and -2 (A1CT and A2CT), simultaneously demonstrating knockdown of one β-arrestin while levels of the other are unaffected. Cells (40-50% confluent) were transfected with 20 nmol of siRNA/10-cm dish or 100 pmol of siRNA/30-mm well using Genesilencer (Gene Therapy Systems), Lipofectamine 2000 (Invitrogen), or N-ter (Sigma) according to the manufacturers' instructions. Assays were performed 48 h after siRNA transfection. Cofilin/β-Arrestin Interactions—Cleared lysates from confluent 15-cm dishes of untransfected MDA-MB-468 cells (lysis buffer: phosphate-buffered saline supplemented with 1% Nonidet P-40 and protease/phosphatase inhibitors) were incubated with 5 μg of rabbit anti-cofilin (Chemicon) prebound to Protein A-agarose for 4 h, analyzed by 15% SDS-PAGE, and blotted with mouse β-arrestin antibody. Alternatively MDA-MB-468 cells were co-transfected with GFP-tagged β-arrestin-1 or -2 and FLAG-cofilin. Cofilin was immunoprecipitated with anti-FLAG followed by SDS-PAGE and Western blotting with anti-FLAG or anti-GFP. CIN/β-Arrestin Interactions—MDA-MB-468 cells, transfected with both FLAG-β-arrestin and Myc-CIN, Myc-CIN or FLAG-β-arrestin alone, or a vector control were treated with 2fAP for 0-90 min and lysed (lysis buffer: 50 mm Hepes, 150 mm NaCl, 10 mm MgCl2, 5% glycerol, and 1% Nonidet P-40 supplemented with protease inhibitors). Cleared lysates were immunoprecipitated with antibody to FLAG or Myc (A14) for 2 h followed by 12% SDS-PAGE and Western blotting with anti-β-arrestin-1, anti-Myc (9E10), or anti-CIN. LIMK/β-Arrestin Interactions—FLAG-β-arrestin-1 and -2 were overexpressed in NIH3T3 cells and immunoprecipitated with anti-FLAG (M2)-agarose. Washed beads from transfected or untransfected cells were mixed with recombinant His-tagged LIMK (amino acids 285-639) for 1 h at room temperature, pelleted, washed, and analyzed by SDS-PAGE followed by Western blotting with anti-FLAG and anti-His6. Alternatively GST-β-arrestin-1, GST-β-arrestin-2, or GST alone were expressed Escherichia coli (strain BL21DE3), protein expression was induced with 0.2 mm isopropyl 1-thio-β-d-galactopyranoside for 4 h, and GST-tagged proteins were purified with glutathione-Sepharose to >85% purity. Some breakdown of both GST-β-arrestin proteins was observed, accounting for the appearance of 29- and 42-45-kDa bands visible by Coomassie. 5 μg each of each GST fusion protein was incubated with 1 or 5 μg of His-tagged LIMK (amino acids 285-639) for 1 h, LIMK-bound proteins were pulled down with cobalt resin, and both bound and unbound proteins were analyzed by SDS-PAGE followed by staining with Coomassie. Actin polymerization assays were performed using an Actin Polymerization Biochem kit (Cytoskeleton Inc.). It has been demonstrated by others that addition of depolymerizing factors such as cytochalasin or actin-depolymerizing factor/cofilin to pyrene-actin filaments at steady state will cause a decrease in fluorescence reflective of depolymerization/severing activity (44Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (837) Google Scholar, 45Cooper J.A. Carraway K.L. Carraway C.A.C. The Cytoskeleton: a Practical Approach. IRL Press, Oxford1992: 47-71Google Scholar, 46Cooper J.A. Schafer D.A. Curr. Opin. Cell Biol. 2000; 12: 97-103Crossref PubMed Scopus (265) Google Scholar, 47Blanchoin L. Pollard T.D. J. Biol. Chem. 1999; 274: 15538-15546Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Although cell extracts will likely contain a myriad of other factors that can affect the actin assembly kinetics, when they are added to filaments that have reached steady state, a decrease in fluorescence reflects the presence of a depolymerizing activity. Cell lysates were prepared from MDA-MB-468 cells and MEFs in lysis buffer (20 mm Hepes, pH 7.0, 4 mm MgCl2, 1 mm ATP, 100 mm KCl, 9mm EGTA, and protease inhibitors). Pyrene-G-actin (10% labeled) was incubated in polymerization buffer (containing 10 mm imidazole, pH 7.5, 2 mm MgCl2, 50 mm KCl, and 1 mm ATP) for 2 h at room temperature, the resulting pyrene-F-actin was diluted to 2 μm (final concentration) in Buffer G (5 mm Tris-HCl, pH 8.0, 0.2 mm CaCl2, 0.2 mm ATP, and 0.5 mm dithiothreitol), and fluorescence was monitored for 3000 s. Cell extracts (40 μg of protein/10 μl of lysis buffer) or 10 μl of lysis buffer alone were added after 1000 s. Severing was monitored by measuring the decrease in fluorescence at 386 nm on a Spex Fluoromax-2 fluorometer. Cofilin has been shown to generate free barbed ends available for elongation (31DesMarais V. Ghosh M. Eddy R. Condeelis J. J. Cell Sci. 2005; 118: 19-26Crossref PubMed Scopus (249) Google Scholar); thus if the fluorescence decrease observed upon addition of PAR-2-stimulated extracts was due to filament severing activity, addition of G-actin should result in increased polymerization. To confirm that a decrease in fluorescence was accompanied by an increase in free barbed ends, at t = 2200 s, pyrene-G-actin (2 μm) was added back to reactions. This also rules out the possibility that the decrease in pyrene fluorescence was due to quenching. Data were collected using Data Max software 2.2 and analyzed using Kaleidagraph Version 4.0. Cleared cell lysates (LIMK lysis buffer: Hepes, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1 mm MgCl2, 1 mm MnCl2, and protease inhibitors) were incubated with anti-LIMK for 8 h at 4 °C. Phosphatase inhibitors were added (1 mm NaF and NaVO4) to cleared lysates just before addition of antibody. Washed beads were incubated in LIMK lysis buffer supplemented with 5 μm cold ATP, 5 μCi of [γ-32P]ATP, and 2 μg of recombinant GST-cofilin at 30 °C for 45 min. Reactions were terminated by transfer of the reaction mixture to 2× Laemmli sample buffer followed by SDS-PAGE, Coomassie staining, and autoradiography. GST-cofilin bands were excised, and incorporated radiolabel was determined by scintillation counting. Immobilized GST-cofilin was phosphorylated with constitutively active His6-LIMK (100 ng of LIMK and 500 ng of GST-cofilin) in LIMK reaction buffer (25 mm Tris, pH 7.2, 0.5 mm EGTA, 250 μm NaVO4, 0.25 mm dithiothreitol, 0.05% β-mercaptoethanol, 0.015% Brij 35, 250 μm cold ATP, 1 μCi of [γ-32P]ATP) for 30 min at 30 °C. GST-cofilin was pelleted, washed, incubated with either lysis buffer alone or lysates from MDA-MB-468 cells treated with 100 nm 2fAP for 0-45 min, and then analyzed by SDS-PAGE followed by autoradiography. Bands were excised and counted in a Beckman scintillation counter. Cells were incubated in physiological salt solution (PSS: 137 mm NaCl, 4.7 mm KCl, 0.56 mm MgCl2, 2 mm CaCl2, 1.0 mm Na2HPO4, 10 mm Hepes, 2.0 mm l-glutamine, and 5.5 mm d-glucose, pH 7.4) containing 0.1% bovine serum albumin and 5 μm Fura-2/AM for 20 min at 37 °C. Cells were washed and mounted in a microincubator containing 1 ml of PSS-bovine serum albumin at 37 °C on the stage of a Nikon inverted microscope (40× objective). Agonists were directly added to the bath. The Ca2+ ionophore, ionomycin, was added at the end of all experiments to determine maximum [Ca2+]. Fluorescence was detected in individual cells using a Nikon charge-coupled device camera and a video microscopy acquisition program (Metafluor). Fluorescence was measured at 340 and 380 nm excitation and 510 nm emission. The ratio of the fluorescence at the two excitation wavelengths, which is proportional to the [Ca2+]i, was determined, and intracellular [Ca2+] was calculated using the Grynkiewicz equation (48Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Average [Ca2+]i values for a range of 10-12 cells were calculated and presented as a single trace. Cells were seeded onto collagen-coated coverslips and allowed to attach overnight. After agonist treatment, cells were fixed in normal buffered formalin and prepared as described previously (9Ge L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Serial sections (1 μm; 100× and 63× objectives) were taken on a Zeiss LSM510 at 1× and 4× zoom. Overlays of four individual Z-sections and enlarged images were obtained using Adobe Photoshop 5.0. All graphs and statistical analyses were performed using Kaleidagraph Version 4.0 or Microsoft Excel 2003. All experiments were performed a minimum of three times. Phosphoprotein levels were normalized to total protein levels before calculating -fold change with respect to untreated controls. For LIMK assays, cpm values were normalized to total LIMK immunoprecipitated and total GST-cofilin levels before calculating -fold change with respect to controls. Analysis of variance and Tukey t tests were used to determine statistical significance of and significant differences between values under different conditions. PAR-2 Promotes Cofilin Dephosphorylation and Filament Severing Activity—Cofilin can be activated by dephosphorylation on Ser3; thus, we investigated whether cofilin phosphorylation was altered in response to the PAR-2 activation using the specific PAR-2 activating peptide 2fAP (49Al Ani B. Saifeddine M. Wijesuriya S.J. Hollenberg M.D. J. Pharmacol. Exp. Ther. 2002; 300: 702-708Crossref PubMed Scopus (62) Google Scholar, 50McGuire J.J. Saifeddine M. Triggle C.R. Sun K. Hollenberg M.D. J. Pharmacol. Exp. Ther. 2004; 309: 1124-1131Crossref PubMed Scopus (117) Google Scholar), inactive reverse 2fAP, or trypsin and probing cell lysates with a phospho (Ser3)-specific cofilin antibody. In the human breast cancer cell line (MDA-MB-468), we observed a robust decrease in phosphorylation as early as 30 s after PAR-2 activation (Fig. 1, A-C). This effect was dose-dependent (Fig. 1B) and was observed in another breast cancer cell line, MDA-MB-231 (not shown). Max" @default.
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• W2039926575 title "β-Arrestin-dependent Regulation of the Cofilin Pathway Downstream of Protease-activated Receptor-2" @default.
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