Calcium Signal-induced Cofilin Dephosphorylation Is Mediated by Slingshot via Calcineurin
2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m411494200
ISSN1083-351X
AutoresYan Wang, Futoshi Shibasaki, Kensaku Mizuno,
Tópico(s)Silk-based biomaterials and applications
ResumoCofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes dephosphorylation in response to extracellular stimuli that elevate intracellular Ca2+ concentrations, signaling mechanisms mediating Ca2+-induced cofilin dephosphorylation have remained unknown. We investigated the role of Slingshot (SSH) 1L, a member of a SSH family of protein phosphatases, in mediating Ca2+-induced cofilin dephosphorylation. The Ca2+ ionophore A23187 and Ca2+-mobilizing agonists, ATP and histamine, induced SSH1L activation and cofilin dephosphorylation in cultured cells. A23187- or histamine-induced SSH1L activation and cofilin dephosphorylation were blocked by calcineurin inhibitors or a dominant-negative form of calcineurin, indicating that calcineurin mediates Ca2+-induced SSH1L activation and cofilin dephosphorylation. Importantly, knockdown of SSH1L expression by RNA interference abolished A23187- or calcineurin-induced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylated SSH1L and increased the cofilin-phosphatase activity of SSH1L in cell-free assays. Based on these findings, we suggest that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent activation of SSH1L. Cofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes dephosphorylation in response to extracellular stimuli that elevate intracellular Ca2+ concentrations, signaling mechanisms mediating Ca2+-induced cofilin dephosphorylation have remained unknown. We investigated the role of Slingshot (SSH) 1L, a member of a SSH family of protein phosphatases, in mediating Ca2+-induced cofilin dephosphorylation. The Ca2+ ionophore A23187 and Ca2+-mobilizing agonists, ATP and histamine, induced SSH1L activation and cofilin dephosphorylation in cultured cells. A23187- or histamine-induced SSH1L activation and cofilin dephosphorylation were blocked by calcineurin inhibitors or a dominant-negative form of calcineurin, indicating that calcineurin mediates Ca2+-induced SSH1L activation and cofilin dephosphorylation. Importantly, knockdown of SSH1L expression by RNA interference abolished A23187- or calcineurin-induced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylated SSH1L and increased the cofilin-phosphatase activity of SSH1L in cell-free assays. Based on these findings, we suggest that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent activation of SSH1L. Actin cytoskeletal reorganization is essential for numerous cell activities, including migration, morphological change, and vesicle transport. The actin-depolymerizing factor/cofilin family proteins are key regulators for actin filament dynamics and reorganization, with potential to bind to actin monomers and filaments and stimulate depolymerization and severance of actin filaments (1.Moon A. Drubin D.G. Mol. Biol. Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (227) Google Scholar, 2.Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (844) Google Scholar, 3.Carlier M.-F. Ressad F. Pantaloni D. J. Biol. Chem. 1999; 274: 33827-33830Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 4.Condeelis J. Trends Cell Biol. 2001; 11: 288-293Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). These activities of actin-depolymerizing factor/cofilin (henceforth referred to as cofilin) are negatively regulated by phosphorylation at Ser-3 and reactivated by dephosphorylation (5.Agnew B.J. Minamide L.S. Bamburg J.R. J. Biol. 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Given the essential role of cofilin in actin filament dynamics, it is important to elucidate signal transduction mechanisms that regulate cofilin phosphorylation and dephosphorylation in order to better understand stimulus-induced actin cytoskeletal remodeling and the cell activities related to it. LIM kinases (LIM kinases 1 and 2) and testicular protein kinases (testicular protein kinases 1 and 2), which specifically phosphorylate cofilin at Ser-3, have been identified as mediators of cofilin phosphorylation (7.Yang N. Higuchi O. Ohashi K. Nagata K. Wada A. Kangawa K. Nishida E. Mizuno K. Nature. 1998; 393: 809-812Crossref PubMed Scopus (1071) Google Scholar, 8.Arber S. Barbayannis F.A. Hanser H. Schneider C. Stanyon C.A. Bernard O. Caroni P. Nature. 1998; 393: 805-809Crossref PubMed Scopus (1165) Google Scholar, 9.Amano T. Tanabe K. Eto T. Narumiya S. Mizuno K. Biochem. J. 2001; 354: 149-159Crossref PubMed Scopus (139) Google Scholar, 10.Toshima J. Toshima J.Y. Amano T. Yang N. 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Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (222) Google Scholar). Thus, cofilin phosphorylation/inactivation is induced by multiple pathways, all of which are dependent on extracellular cues. As for phosphatases responsible for cofilin dephosphorylation, genetic and biochemical analyses have led to identification of a Slingshot (SSH) 1The abbreviations used are: SSH, Slingshot; CnA, calcineurin A β-isotype; HA, hemagglutinin; P-cofilin, Ser-3-phosphorylated cofilin; siRNA, small interfering RNA; WT, wild-type. family of protein phosphatases (SSH in Drosophila and SSH1L, SSH2L, and SSH3L in mammals), which can specifically dephosphorylate and reactivate an inactive Ser-3-phosphorylated cofilin (P-cofilin), both in vitro and in vivo (16.Niwa R. Nagata-Ohashi K. Takeichi M. Mizuno K. Uemura T. Cell. 2002; 108: 233-246Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar, 17.Ohta Y. Kousaka K. Nagata-Ohashi K. Ohashi K. Muramoto A. Shima Y. Niwa R. Uemura T. Mizuno K. Genes Cells. 2003; 8: 811-824Crossref PubMed Scopus (97) Google Scholar, 18.Endo M. Ohashi K. Sasaki Y. Goshima Y. Niwa R. Uemura T. Mizuno K. J. Neurosci. 2003; 23: 2527-2537Crossref PubMed Google Scholar, 19.Kaji N. Ohashi K. Shuin M. Niwa R. Uemura T. Mizuno K. J. Biol. Chem. 2003; 278: 33450-33455Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Cofilin dephosphorylation is induced in response to various extracellular stimuli in many different cell types (1.Moon A. Drubin D.G. Mol. Biol. Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (227) Google Scholar, 2.Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (844) Google Scholar, 3.Carlier M.-F. Ressad F. Pantaloni D. J. Biol. Chem. 1999; 274: 33827-33830Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 4.Condeelis J. 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Meuer S.C. Samstag Y. Eur. J. Immunol. 2000; 30: 892-899Crossref PubMed Scopus (64) Google Scholar, 27.Ambach A. Saunus J. Konstandin M. Wesselborg S. Meuer S.C. Samstag Y. Eur. J. Immunol. 2000; 30: 3422-3431Crossref PubMed Scopus (140) Google Scholar, 28.Zhan Q. Bamburg J.R. Badway J.A. Cell Motil. Cytoskeleton. 2003; 54: 1-15Crossref PubMed Scopus (37) Google Scholar, 29.Nishita M. Wang Y. Tomizawa C. Suzuki A. Niwa R. Uemura T. Mizuno K. J. Biol. Chem. 2004; 279: 7193-7198Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 30.Nagata-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). Although multiple signaling pathways, including those involving Ca2+, cAMP, and phosphoinositide 3-kinase, have been proposed for stimulus-induced cofilin dephosphorylation (20.Davidson M.M. Haslam R.J. Biochem. J. 1994; 301: 41-47Crossref PubMed Scopus (74) Google Scholar, 21.Okada K. Takano-Ohmuro H. Obinata T. Abe H. Exp. Cell Res. 1996; 227: 116-122Crossref PubMed Scopus (56) Google Scholar, 22.Takuma T. Ichida T. Yokoyama N. Tamura S. Obinata T. J. Biochem. (Tokyo). 1996; 120: 35-41Crossref PubMed Scopus (27) Google Scholar, 23.Meberg P.J. Ono S. Minamide L.S. Takahashi M. Bamburg J.R. Cell Motil. Cytoskeleton. 1998; 39: 172-190Crossref PubMed Scopus (217) Google Scholar, 24.Birkenfeld J. Kartmann B. Betz H. Roth D. Biochem. Biophys. Res. Commun. 2001; 286: 493-498Crossref PubMed Scopus (14) Google Scholar, 25.Suzuki K. Yamaguchi T. Tanaka T. Kawanishi T. Nishimaki-Mogami T. Yamamoto K. Tsuji T. Irimura T. Hayakawa T. Takahashi A. J. Biol. Chem. 1995; 270: 19551-19556Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 26.Lee K.H. Meuer S.C. Samstag Y. Eur. J. Immunol. 2000; 30: 892-899Crossref PubMed Scopus (64) Google Scholar, 27.Ambach A. Saunus J. Konstandin M. Wesselborg S. Meuer S.C. Samstag Y. Eur. J. Immunol. 2000; 30: 3422-3431Crossref PubMed Scopus (140) Google Scholar, 28.Zhan Q. Bamburg J.R. Badway J.A. Cell Motil. Cytoskeleton. 2003; 54: 1-15Crossref PubMed Scopus (37) Google Scholar), neither downstream signaling pathways nor the roles of SSHs in stimulus-induced cofilin dephosphorylation have been determined. We recently showed that SSH1L is activated downstream of phosphoinositide 3-kinase in insulin-stimulated cells, but the signaling pathway linking phosphoinositide 3-kinase and SSH1L has remained unknown (29.Nishita M. Wang Y. Tomizawa C. Suzuki A. Niwa R. Uemura T. Mizuno K. J. Biol. Chem. 2004; 279: 7193-7198Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). We also showed that the cofilin-phosphatase activity of SSH1L is increased by association with actin filaments and suppressed by 14-3-3 proteins (30.Nagata-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). However, the mechanisms regulating SSH1L activity through F-actin and 14-3-3 proteins are not well understood. In the present study, we investigated the signaling mechanism of Ca2+ signal-induced cofilin dephosphorylation. Previous studies reported that elevation of the intracellular Ca2+ concentration stimulates cofilin dephosphorylation in a variety of cell types (20.Davidson M.M. Haslam R.J. Biochem. J. 1994; 301: 41-47Crossref PubMed Scopus (74) Google Scholar, 21.Okada K. Takano-Ohmuro H. Obinata T. Abe H. Exp. Cell Res. 1996; 227: 116-122Crossref PubMed Scopus (56) Google Scholar, 22.Takuma T. Ichida T. Yokoyama N. Tamura S. Obinata T. J. Biochem. (Tokyo). 1996; 120: 35-41Crossref PubMed Scopus (27) Google Scholar, 23.Meberg P.J. Ono S. Minamide L.S. Takahashi M. Bamburg J.R. Cell Motil. Cytoskeleton. 1998; 39: 172-190Crossref PubMed Scopus (217) Google Scholar, 24.Birkenfeld J. Kartmann B. Betz H. Roth D. Biochem. Biophys. Res. Commun. 2001; 286: 493-498Crossref PubMed Scopus (14) Google Scholar), and this dephosphorylation is blocked by inhibitors of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (also called protein phosphatase 2B), thus indicating that calcineurin is involved in Ca2+-induced cofilin dephosphorylation (23.Meberg P.J. Ono S. Minamide L.S. Takahashi M. Bamburg J.R. Cell Motil. Cytoskeleton. 1998; 39: 172-190Crossref PubMed Scopus (217) Google Scholar, 31.Klee C.B. Ren H. Wang X. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). However, it remained to be determined whether or not calcineurin directly dephosphorylates cofilin or merely mediates cofilin dephosphorylation in cells through activation of a cofilin-specific phosphatase, such as SSH1L. Here we now provide evidence that Ca2+-induced cofilin dephosphorylation is mediated by SSH1L via calcineurin. Materials—A23187, histamine, cyclosporin A, FK506, cypermethrin, calmodulin, and recombinant active calcineurin Aα containing calcineurin B subunit were purchased from Calbiochem, and okadaic acid was purchased from Wako (Osaka, Japan). Mouse monoclonal antibody against Myc epitope (9E10) and rat monoclonal antibody against hemagglutinin (HA) epitope (3F10) were purchased form Roche Diagnostics. Rabbit polyclonal antibodies to P-cofilin, cofilin, and SSH1L were prepared as described previously (10.Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (222) Google Scholar, 19.Kaji N. Ohashi K. Shuin M. Niwa R. Uemura T. Mizuno K. J. Biol. Chem. 2003; 278: 33450-33455Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). An anti-β-actin monoclonal antibody (AC-15) was purchased from Sigma. Fluorescein isothiocyanate-conjugated anti-rat IgG antibody and rhodamine-conjugated anti-rabbit IgG antibody were purchased from Chemicon (Temecula, CA). Plasmid Construction—Expression plasmids coding for Myc epitope-tagged wild-type SSH1L (SSH1L(WT)) and phosphatase-dead SSH1L (SSH1L(CS)) were constructed as described previously (16.Niwa R. Nagata-Ohashi K. Takeichi M. Mizuno K. Uemura T. Cell. 2002; 108: 233-246Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar, 17.Ohta Y. Kousaka K. Nagata-Ohashi K. Ohashi K. Muramoto A. Shima Y. Niwa R. Uemura T. Mizuno K. Genes Cells. 2003; 8: 811-824Crossref PubMed Scopus (97) Google Scholar). Plasmids for HA epitope-tagged calcineurin A β-isotype (CnA), its constitutively active (ΔCnA) and dominant-negative (ΔCnA(H160Q)) mutants, and calcineurin B were constructed as described previously (31.Klee C.B. Ren H. Wang X. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 32.Shibasaki F. Price E.R. Milan D. McKeon F. Nature. 1996; 382: 370-373Crossref PubMed Scopus (434) Google Scholar). pSUPER vector for small interfering RNA (siRNA) was provided by Dr. R. Agami (The Netherlands Cancer Institute, Amsterdam, The Netherlands) (33.Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar). An SSH1L siRNA plasmid (pSUPER-SSH1L) that targets the human SSH1L mRNA sequence (TCGTCACCCAAGAAAGATA) was constructed as described previously (29.Nishita M. Wang Y. Tomizawa C. Suzuki A. Niwa R. Uemura T. Mizuno K. J. Biol. Chem. 2004; 279: 7193-7198Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). A mutated SSH1L siRNA plasmid, pSUPER-SSH1L(mt), was constructed by substituting two bases in the target sequence (TCTTCCCCCAAGAAAGATA). Cell Culture and Transfection—293T and HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. 293T and HeLa cells were transfected with expression plasmids using FuGENE 6 transfection reagent (Roche Applied Science) and Lipofectamine (Invitrogen), respectively. Cells were used for assays after being in culture for 30–44 h. Detection of the Level of P-cofilin—Serum-starved 293T or HeLa cells were washed twice with phosphate-buffered saline, harvested, and lysed with lysis buffer (50 mm HEPES, pH 7.4, 1% Nonidet P-40, 10% glycerol, 1 mm EDTA, 1 mm dithiothreitol, and 10 μg/ml leupeptin). After incubation on ice for 30 min, lysates were incubated in Laemmli's sample buffer (50 mm Tris-HCl, pH 6.8, 10% glycerol, 1 mm dithiothreitol, 1% SDS, and 0.002% bromphenol blue) for 5 min at 95 °C, and aliquots were separated by SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membranes. The membranes were analyzed by immunoblotting using anti-P-cofilin and anti-cofilin antibodies. Immunoblot analysis was performed as described previously (15.Ohashi K. Nagata K. Maekawa M. Ishizaki T. Narumiya S. Mizuno K. J. Biol. Chem. 2000; 275: 3577-3582Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). In Vitro Phosphatase Assay of SSH1L—Cofilin-His6 expressed in Vero cells was purified and used as a substrate for in vitro phosphatase reactions. 293T or HeLa cells were lysed in phosphatase buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 5% glycerol, 1 mm dithiothreitol, and 10 μg/ml leupeptin). SSH1L was immunoprecipitated with anti-SSH1L antibody and incubated with purified cofilin-His6 for 2 h at 30 °C. Reaction mixtures were analyzed by immunoblotting using antibodies to cofilin, P-cofilin, and SSH1L. To examine the effect of calcineurin on SSH1L activity in cell-free assays, lysates of 293T cells were immunoprecipitated with anti-SSH1L antibody or control IgG. The precipitates were incubated for 1 h at 30 °C with 7.5 units/μl recombinant active calcineurin Aα containing calcineurin B subunit, 14 μg/ml calmodulin, and 6.3 μg/ml cofilin-His6 in 20 μl of reaction buffer (50 mm HEPES, pH 7.4, 0.1 mm CaCl2, 0.5 mm EDTA, 3 mm MgCl2, 100 mm NaCl, 1 mm dithiothreitol, 0.5 mg/ml bovine serum albumin, and 1 μg/ml leupeptin) and then further incubated for 30 min at 30 °C in reaction buffer containing 0.25 mg/ml F-actin. Reaction mixtures were separated by SDS-PAGE and analyzed by immunoblotting using antibodies to cofilin, P-cofilin, and SSH1L. Dephosphorylation of SSH1L by Calcineurin in Cell-free Assays— Lysates of 293T cells transfected with plasmids for Myc-SSH1L were immunoprecipitated with anti-Myc antibody or control IgG. The precipitates were incubated with recombinant active calcineurin Aα and B and calmodulin, as described above. Phosphatase inhibitor (5 mm pyrophosphate) was preincubated with active calcineurin for 15 min at 4 °C. Reaction mixtures were run on SDS-PAGE, and the amounts of phosphorylated SSH1L and total SSH1L were analyzed by staining the gel with Pro-Q Diamond phosphoprotein gel stain kit (Molecular Probes) and Coomassie Brilliant Blue, respectively. Cell Staining—HeLa cells were fixed with 4% formaldehyde in phosphate-buffered saline for 20 min and permeabilized with 100% methanol for 5 min. After blocking with 1% bovine serum albumin in phosphate-buffered saline, cells were incubated with 3F10 rat anti-HA monoclonal antibody and rabbit anti-P-cofilin antibodies, followed by incubation with fluorescein isothiocyanate-conjugated anti-rat IgG antibody and rhodamine-conjugated anti-rabbit IgG antibody. Coverslips were washed with phosphate-buffered saline and mounted on glass slides. Images were obtained using a Leica DMLB fluorescence microscope. Ca2+Ionophore Stimulates Cofilin Dephosphorylation and SSH1L Activation—It was reported that cofilin undergoes dephosphorylation in response to extracellular stimuli that increase the intracellular Ca2+ concentration (20.Davidson M.M. Haslam R.J. Biochem. J. 1994; 301: 41-47Crossref PubMed Scopus (74) Google Scholar, 21.Okada K. Takano-Ohmuro H. Obinata T. Abe H. Exp. Cell Res. 1996; 227: 116-122Crossref PubMed Scopus (56) Google Scholar, 22.Takuma T. Ichida T. Yokoyama N. Tamura S. Obinata T. J. Biochem. (Tokyo). 1996; 120: 35-41Crossref PubMed Scopus (27) Google Scholar, 23.Meberg P.J. Ono S. Minamide L.S. Takahashi M. Bamburg J.R. Cell Motil. Cytoskeleton. 1998; 39: 172-190Crossref PubMed Scopus (217) Google Scholar, 24.Birkenfeld J. Kartmann B. Betz H. Roth D. Biochem. Biophys. Res. Commun. 2001; 286: 493-498Crossref PubMed Scopus (14) Google Scholar). To explore signaling pathways of Ca2+-induced cofilin dephosphorylation, we first examined the effects of the Ca2+ ionophore A23187 on levels of cofilin phosphorylation in cultured cells. Serum-starved 293T or HeLa cells were exposed to 5 μm A23187 for different time periods, and cell lysates were analyzed by immunoblotting with an anti-P-cofilin antibody that specifically recognizes P-cofilin and an anti-cofilin antibody that recognizes both phosphorylated and non-phosphorylated forms of cofilin (10.Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (222) Google Scholar). Stimulation of 293T or HeLa cells with A23187 significantly decreased the level of P-cofilin in a time-dependent manner, without affecting the total cofilin level (Fig. 1A). A23187-induced cofilin dephosphorylation was also observed in WI-38 human lung fibroblasts (data not shown). These findings suggest that the increase in intracellular Ca2+ concentrations induces cofilin dephosphorylation in various types of cells. We next analyzed changes in cofilin-phosphatase activity of endogenous SSH1L after stimulating the cells with A23187. Serum-starved 293T or HeLa cells were incubated with or without 5 μm A23187 for 10 min, cell lysates were prepared, and SSH1L was immunoprecipitated with an anti-SSH1L antibody. The immunoprecipitates were subjected to an in vitro phosphatase assay, using a recombinant phosphorylated cofilin-His6 substrate. Cofilin-phosphatase activity was measured by the decrease in P-cofilin immunoreactivity. In both 293T and HeLa cells, the cofilin-phosphatase activity of SSH1L significantly increased after A23187 stimulation (Fig. 1B). These results suggest that Ca2+ signaling induces both SSH1L activation and cofilin dephosphorylation. Ca2+-mobilizing Agents Induce Cofilin Dephosphorylation and SSH1L Activation—Extracellular ATP and histamine are known to elevate the intracellular Ca2+ concentration by mobilizing Ca2+ from internal stores in 293T and HeLa cells, respectively (34.Aguiari G. Campanella M. Manzati E. Pinton P. Banzi M. Moretti S. Piva R. Rizzuto R. del Senno L. Biochem. Biophys. Res. Commun. 2003; 301: 657-664Crossref PubMed Scopus (23) Google Scholar). We asked whether these Ca2+-mobilizing agents would induce cofilin dephosphorylation and SSH1L activation in these cells. The level of P-cofilin significantly decreased after exposure of 293T cells to 50 μm ATP (Fig. 2A, top panel). In vitro phosphatase assay showed that the cofilin-phosphatase activity of SSH1L in 293T cells increased after ATP stimulation (Fig. 2A, bottom panel). In a similar manner, stimulation of HeLa cells with 10 mm histamine induced both cofilin dephosphorylation and SSH1L activation (Fig. 2B). Therefore extracellular Ca2+-mobilizing agents do have the potential to induce cofilin dephosphorylation and SSH1L activation. Calcineurin Inhibitors Block Ca2+-induced Cofilin Dephosphorylation and SSH1L Activation—A23187-induced cofilin dephosphorylation was reported to be blocked by calcineurin inhibitors, which suggested that calcineurin is involved in the Ca2+-induced cofilin dephosphorylation (23.Meberg P.J. Ono S. Minamide L.S. Takahashi M. Bamburg J.R. Cell Motil. Cytoskeleton. 1998; 39: 172-190Crossref PubMed Scopus (217) Google Scholar). However, it is unclear whether or not calcineurin directly dephosphorylates cofilin. We examined the effects of calcineurin inhibitors on A23187- or histamine-induced cofilin dephosphorylation and SSH1L activation in HeLa cells. When HeLa cells were preincubated with calcineurin inhibitors (10 μm cyclosporin A, 50 nm FK506, or 100 nm cypermethrin) and then treated with 5 μm A23187 for 10 min, A23187-induced cofilin dephosphorylation was almost completely blocked in the presence of any one of these calcineurin inhibitors (Fig. 3A, top panel). Interestingly, A23187-induced SSH1L activation was also suppressed by calcineurin inhibitors (Fig. 3A, bottom panel), which indicates that calcineurin is required for the Ca2+-induced SSH1L activation. Both histamine-induced cofilin dephosphorylation and SSH1L activation in HeLa cells were inhibited by pretreatment of the cells with FK506 (Fig. 3B). These findings suggest that Ca2+-induced cofilin dephosphorylation and SSH1L activation are dependent on calcineurin activity and that calcineurin functions upstream of SSH1L. In addition, pretreatment of cells with 1 μm okadaic acid (an inhibitor of protein phosphatases type 1 and 2A) had no apparent effect on histamine-induced cofilin dephosphorylation and SSH1L activation (Fig. 3C), which suggests that neither protein phosphatase type 1 nor 2A is involved in Ca2+-induced cofilin dephosphorylation and SSH1L activation. Expression of Dominant-negative Calcineurin Suppresses A23187-induced Cofilin Dephosphorylation and SSH1L Activation—To further examine the role of calcineurin in Ca2+-induced cofilin dephosphorylation and SSH1L activation, we overexpressed either HA-tagged wild-type calcineurin A (CnA(WT)) or its dominant-negative form (ΔCnA(H160Q)) in HeLa cells and analyzed changes in the levels of P-cofilin and SSH1L activity before and after A23187 treatment. Expression of CnA(WT) alone had no apparent effect, unless cells had been exposed to agents that increased the intracellular Ca2+ concentration (32.Shibasaki F. Price E.R. Milan D. McKeon F. Nature. 1996; 382: 370-373Crossref PubMed Scopus (434) Google Scholar). A23187-induced cofilin dephosphorylation and SSH1L activation were suppressed in cells expressing ΔCnA(H160Q), but not in cells expressing CnA(WT) (Fig. 4). These data further indicate that calcineurin activity is required for Ca2+-induced SSH1L activation and cofilin dephosphorylation. Phosphatase-inactive SSH1L Suppresses A23187-induced Cofilin Dephosphorylation—To examine the role of SSH1L in Ca2+-induced cofilin dephosphorylation, we overexpressed either SSH1L(WT) or SSH1L(CS) in HeLa cells and analyzed changes in P-cofilin levels before and after A23187 stimulation. In cells expressing SSH1L(WT), the level of P-cofilin decreased significantly even before A23187 stimulation and declined further after stimulation (Fig. 5A). In contrast, in cells expressing SSH1L(CS), a phosphatase-inactive Slingshot-1L mutant in which catalytic Cys is replaced by Ser, A23187-induced cofilin dephosphorylation was significantly suppressed (Fig. 5A). Similar results were obtained when CnA(WT) was co-expressed with either SSH1L(WT) or SSH1L(CS) in HeLa cells. A23187-induced cofilin dephosphorylation in CnA(WT)-expressing cells was stimulated by co-expression of SSH1L(WT) and inhibited by SSH1L(CS) (Fig. 5B, compare lanes 3, 6, and 9). These findings suggest that the phosphatase activity of SSH1L is critical for Ca2+/calcineurin-induced cofilin dephosphorylation and that SSH1L(CS) acts as a dominant-negative mutant. Suppression of SSH1L Expression by siRNA Inhibits A23187-induced Cofilin Dephosphorylation—To confirm that SSH1L mediates Ca2+/calcineurin-induced cofilin dephosphorylation, expression of endogenous SSH1L in HeLa cells was suppressed by transfection of a pSUPER siRNA expression plasmid, pSUPER-SSH1L, which directs the synthesis of an siRNA targeting human SSH1L (29.Nishita M. Wang Y. Tomizawa C. Suzuki A. Niwa R. Uemura T. Mizuno K. J. Biol. Chem. 2004; 279: 7193-7198Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). An empty pSUPER vector and siRNA plasmid mutated in the SSH1L target sequence, pSUPER-SSH1L(mt), were transfected as control experiments. Immunoblot analysis revealed that transfection with pSUPER-SSH1L significantly reduced the level of endogenous SSH1L expression, whereas control vector or mutated siRNA plasmid did not suppress it (Fig. 6A). HeLa cells were co-transfected with CnA(WT) and siRNA plasmids, and changes in P-cofilin levels in cells were analyzed before and after A23187 stimulation. In cells transfected with control pSUPER vector or pSUPER-SSH1L(mt), A23187 stimulation induced cofilin dephosphorylation (Fig. 6B, lanes 3 and 9). In contrast, in cells transfected with pSUPER-SSH1L, A23187-induced cofilin dephosphorylation was significantly inhibited (Fig. 6B, lane 6). These observations further suggest that SSH1L plays a critical role in Ca2+/calcineurin-induced cofilin dephosphorylation. Suppression of SSH1L Expression by siRNA Inhibits Calcineurin-induced Cofilin Dephosphorylation—We further examined the effects of SSH1L RNA interference on calcineurin-induced cofilin dephosphorylation by staining cells with an anti-P-cofilin antibody. HeLa cells transfected with plasmids coding for HA-tagged constitutively active calcineurin A and calcineurin B (ΔCnA/B) with or without pSUPER-SSH1L siRNA plasmids were cultured for 44 h and then co-stained with anti-HA and anti-P-cofilin antibodies. As shown in the left panels of Fig. 7, the P-cofilin level significantly decreased in cells expressing ΔCnA/B (indicated by arrowheads), compared with the levels in neighboring non-expressing cells. In contrast, the P-cofilin levels in cells co-transfected with ΔCnA/B and pSUPER-SSH1L were similar to those in surrounding cells (Fig. 7, right panels), which indicates that SSH1L is required for ΔCnA/B-induced cofilin dephosphorylation. These observations strongly suggest that calcineurin induces cofilin dephosphorylation via SSH1L activation. Calcineurin Dephosphorylates and Activates SSH1L in Cell-free Assays—We next examined whether calcineurin has the potential to dephosphorylate and activate SSH1L in cell-free assays. Lysates of 293T cells transfected with plasmids for Myc-SSH1L were immunoprecipitated with anti-Myc antibody or control IgG, the precipitates were incubated in the presence or absence of recombinant active calcineurin, and the levels of SSH1L phosphorylation were analyzed (Fig. 8A). The amounts of phosphorylated SSH1L (P-SSH1L) and total SSH1L were measured by Pro-Q Diamond phosphoprotein staining and Coomassie Brilliant Blue staining, respectively. Pro-Q Diamond staining revealed that Myc-SSH1L was phosphorylated in 293T cells (Fig. 8A, lane 3) and dephosphorylated in vitro by treatment with active calcineurin (Fig. 8A, lane 5). Calcineurin-catalyzed dephosphorylation was blocked by pretreatment of calcineurin with pyrophosphate, an inhibitor of phosphatases including calcineurin (Fig. 8A, lane 6). These results suggest that SSH1L is a phosphoprotein in cultured cells and that calcineurin has the potential to dephosphorylate SSH1L in cell-free assays. The slight increase in the P-SSH1L level by pyrophosphate may be due to the inhibitory effect on SSH1L autodephosphorylation (Fig. 8A, lane 4). We also examined the effect of active calcineurin on the cofilin-phosphatase activity of SSH1L in cell-free assays. SSH1L purified from 293T cells by immunoprecipitation was incubated with or without recombinant active calcineurin and subjected to the in vitro cofilin-phosphatase assay (Fig. 8B). Incubation of SSH1L with calcineurin significantly increased the cofilin-phosphatase activity of SSH1L (Fig. 8B, lanes 3 and 4). In control experiments, calcineurin alone had no apparent effect on the P-cofilin level (Fig. 8B, lane 2). These results suggest that calcineurin has the potential to stimulate the cofilin-phosphatase activity of SSH1L, but not to catalyze cofilin dephosphorylation itself, in cell-free assays. These data, taken together with the data of Fig. 8A, indicate that calcineurin appears to activate SSH1L by dephosphorylation. Cofilin plays an essential role in regulating actin filament dynamics. Its actin-depolymerizing and -severing activities are inhibited by phosphorylation at Ser-3 by LIM kinases and testicular protein kinases and reactivated by dephosphorylation by SSH family phosphatases (7.Yang N. Higuchi O. Ohashi K. Nagata K. Wada A. Kangawa K. Nishida E. Mizuno K. Nature. 1998; 393: 809-812Crossref PubMed Scopus (1071) Google Scholar, 8.Arber S. Barbayannis F.A. Hanser H. Schneider C. Stanyon C.A. Bernard O. Caroni P. Nature. 1998; 393: 805-809Crossref PubMed Scopus (1165) Google Scholar, 9.Amano T. Tanabe K. Eto T. Narumiya S. Mizuno K. Biochem. J. 2001; 354: 149-159Crossref PubMed Scopus (139) Google Scholar, 10.Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (222) Google Scholar, 11.Toshima J. Toshima J.Y. Takeuchi K. Mori R. Mizuno K. J. Biol. 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Genes Cells. 2003; 8: 811-824Crossref PubMed Scopus (97) Google Scholar). Although a variety of extracellular stimuli induce cofilin dephosphorylation, the signaling pathways that lead to cofilin dephosphorylation have not been well understood. In the present study, we provided evidence that Ca2+ signal-induced cofilin dephosphorylation is mediated by consecutive activation of two protein phosphatases, calcineurin and SSH1L. We showed that Ca2+ ionophore and Ca2+-mobilizing agents induced SSH1L activation and cofilin dephosphorylation, both of which were suppressed by inhibition of calcineurin. These data indicate that Ca2+-induced SSH1L activation and cofilin dephosphorylation are mediated by calcineurin. Importantly, knockdown of endogenous SSH1L expression by siRNA abolished Ca2+- or calcineurin-induced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylated SSH1L and stimulated cofilin-phosphatase activity of SSH1L in cell-free assays, but it had no apparent activity to catalyze cofilin dephosphorylation directly. Taken together, these findings suggest that SSH1L is essential for Ca2+-induced cofilin dephosphorylation as a downstream effector of calcineurin and that calcineurin stimulates cofilin dephosphorylation apparently only through SSH1L activation. Thus, we propose a novel signaling cascade of Ca2+-calcineurin-SSH1L-cofilin for Ca2+ signal-induced cofilin dephosphorylation and activation. The precise mechanism of calcineurin-mediated SSH1L activation is still unclear. It is important to identify the residue(s) of SSH1L that is phosphorylated in resting cells but becomes dephosphorylated by calcineurin in response to stimulation of the Ca2+-induced signaling pathway. SSH1L activity is inhibited by association with 14-3-3 proteins, in a manner dependent on the phosphorylation of SSH1L serine residues (30.Nagata-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), and it is possible that calcineurin stimulates SSH1L activity by dephosphorylating the serine residues that are involved in 14-3-3 binding. In the case of Ca2+-induced apoptosis, calcineurin-mediated dephosphorylation of BAD causes its dissociation from 14-3-3 and translocation to mitochondria to inhibit Bcl-xL (35.Wang H.-G. Pathan N. Ethell I.M. Krajewski S. Yamaguchi Y. Shibasaki F. McKeon F. Bobo T. Franke T.F. Reed J.C. Science. 1999; 284: 339-343Crossref PubMed Scopus (967) Google Scholar). Additional studies are necessary to determine whether calcineurin induces SSH1L activation through a similar mechanism. Ca2+ is a versatile intracellular signal mediator that can regulate many different cellular processes, including those related to actin cytoskeletal rearrangement. Ca2+-mediated cofilin dephosphorylation has been observed in various cell types and is thought to contribute to various cell responses, such as thrombin-induced platelet aggregation (20.Davidson M.M. Haslam R.J. Biochem. J. 1994; 301: 41-47Crossref PubMed Scopus (74) Google Scholar), chemotactic peptide-induced neutrophil migration (28.Zhan Q. Bamburg J.R. Badway J.A. Cell Motil. Cytoskeleton. 2003; 54: 1-15Crossref PubMed Scopus (37) Google Scholar), and nicotine-induced noradrenalin secretion by adrenal chromaffin cells (24.Birkenfeld J. Kartmann B. Betz H. Roth D. Biochem. Biophys. Res. Commun. 2001; 286: 493-498Crossref PubMed Scopus (14) Google Scholar). Because cofilin appears to play an essential role in actin filament dynamics and remodeling, Ca2+-induced SSH1L activation and cofilin dephosphorylation/activation must represent an important signaling pathway supporting stimulus-induced actin cytoskeletal changes and related cell responses. In neurons, intracellular Ca2+ signals play important roles in regulating growth cone motility and guidance (36.Henley J. Poo M.-m. Trends Cell Biol. 2004; 14: 320-330Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Local Ca2+ signals can cause growth cone steering responses, with higher Ca2+ signals mediating attraction and lower Ca2+ signals mediating repulsion (37.Hong K. Nishiyama M. Henley J. Tessier-Lavigne M. Poo M.-m. Nature. 2000; 403: 93-98Crossref PubMed Scopus (311) Google Scholar). Based on findings that both cofilin and SSH1L stimulate growth cone motility and extension (18.Endo M. Ohashi K. Sasaki Y. Goshima Y. Niwa R. Uemura T. Mizuno K. J. Neurosci. 2003; 23: 2527-2537Crossref PubMed Google Scholar, 38.Meberg P.J. Bamburg J.R. J. Neurosci. 2000; 20: 2459-2469Crossref PubMed Google Scholar), local activation of the Ca2+-SSH1L-cofilin signaling pathway appears to stimulate the directional steering of growth cones. In addition, Ca2+ and calcineurin are known to regulate neuronal plasticity and memory formation in the central nervous system (39.Groth R.D. Dunbar R.L. Mermelstein P.G. Biochem. Biophys. Res. Commun. 2003; 311: 1159-1171Crossref PubMed Scopus (135) Google Scholar). Modulation of actin cytoskeletal dynamics in dendritic spines by cofilin phosphorylation and dephosphorylation contributes to spine structure, persistence of long-term potentiation, and synaptic plasticity (40.Meng Y. Zhang Y. Tregoubov V. Janus C. Cruz L. Jackson M. Lu W.Y. MacDonald J.F. Falles D.L. Jia Z. Neuron. 2002; 35: 121-133Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 41.Fukazawa Y. Saitoh Y. Ozawa F. Ohta Y. Mizuno K. Inokuchi K. Neuron. 2003; 38: 447-460Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). Thus, Ca2+/calcineurin-induced SSH1L activation may be one of the mechanisms by which Ca2+/calcineurin regulates neuronal actin cytoskeletal dynamics and hence modulates synaptic function and neuronal plasticity. Various signaling pathways are involved in stimulus-induced cofilin dephosphorylation. In insulin-stimulated cells, phosphoinositide 3-kinase and its product, phosphatidylinositol-3,4,5-trisphosphate, are involved in SSH1L activation and cofilin dephosphorylation (29.Nishita M. Wang Y. Tomizawa C. Suzuki A. Niwa R. Uemura T. Mizuno K. J. Biol. Chem. 2004; 279: 7193-7198Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In other types of cells, cAMP and protein kinase C signals stimulate cofilin dephosphorylation (1.Moon A. Drubin D.G. Mol. Biol. Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (227) Google Scholar, 2.Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (844) Google Scholar, 21.Okada K. Takano-Ohmuro H. Obinata T. Abe H. Exp. Cell Res. 1996; 227: 116-122Crossref PubMed Scopus (56) Google Scholar, 22.Takuma T. Ichida T. Yokoyama N. Tamura S. Obinata T. J. Biochem. (Tokyo). 1996; 120: 35-41Crossref PubMed Scopus (27) Google Scholar, 23.Meberg P.J. Ono S. Minamide L.S. Takahashi M. Bamburg J.R. Cell Motil. Cytoskeleton. 1998; 39: 172-190Crossref PubMed Scopus (217) Google Scholar). In contrast to the Ca2+ signal, cAMP- or protein kinase C signal-induced cofilin dephosphorylation is insensitive to calcineurin or calmodulin inhibitors (23.Meberg P.J. Ono S. Minamide L.S. Takahashi M. Bamburg J.R. Cell Motil. Cytoskeleton. 1998; 39: 172-190Crossref PubMed Scopus (217) Google Scholar, 28.Zhan Q. Bamburg J.R. Badway J.A. Cell Motil. Cytoskeleton. 2003; 54: 1-15Crossref PubMed Scopus (37) Google Scholar), which indicates the existence of calcineurin-independent signaling pathways for cofilin dephosphorylation. Additional studies are required to determine whether SSH1L, other members of a SSH family (e.g. SSH2L and SSH3L), or other more general classes of protein phosphatases, such as protein phosphatase type 1 and 2A (27.Ambach A. Saunus J. Konstandin M. Wesselborg S. Meuer S.C. Samstag Y. Eur. J. Immunol. 2000; 30: 3422-3431Crossref PubMed Scopus (140) Google Scholar), are involved in cAMP- and protein kinase C-induced cofilin dephosphorylation. Indeed, cofilin dephosphorylation/activation appears to be one of the important and convergent points in a cell signaling network through which a variety of extracellular stimuli regulate actin cytoskeletal dynamics and organization. We thank Dr. R. Agami for providing pSUPER plasmid and Drs. R. Niwa, T. Uemura, K. Nagata-Ohashi, M. Nishita, S. Kurita, and M. Ohara for helpful comments.
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