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W2028497798 abstract "Cofilin plays an essential role in actin filament dynamics and membrane protrusion in motile cells. Cofilin is inactivated by phosphorylation at Ser-3 by LIM kinase and reactivated by dephosphorylation by cofilin-phosphatase Slingshot (SSH). Although cofilin is dephosphorylated in response to various extracellular stimuli, signaling pathways regulating SSH activation and cofilin dephosphorylation have remained to be elucidated. Here we show that insulin stimulates the phosphatase activity of Slingshot-1L (SSH1L) and cofilin dephosphorylation in cultured cells, in a manner dependent on phosphoinositide 3-kinase (PI3K) activity. Consistent with this, the level of Ser-3-phosphorylated cofilin is increased in PTEN (phosphatase and tensin homolog deleted in chromosome 10)-overexpressing cells and decreased in PTEN-deficient cells. Insulin induced the accumulation of SSH1L and active Akt (a downstream effector of PI3K), together with a PI3K product phosphatidylinositol 3,4,5-trisphosphate, onto membrane protrusions. Cofilin, but not Ser-3-phosphorylated cofilin, accumulated in membrane protrusions in insulin-stimulated cells, indicating that cofilin is dephosphorylated in these areas. Finally, suppression of SSH1L expression by RNA interference abolished insulin-induced cofilin dephosphorylation and the membrane protrusion. These findings suggest that SSH1L is activated downstream of PI3K and plays a critical role in insulin-induced membrane protrusion by dephosphorylating and activating cofilin. Cofilin plays an essential role in actin filament dynamics and membrane protrusion in motile cells. Cofilin is inactivated by phosphorylation at Ser-3 by LIM kinase and reactivated by dephosphorylation by cofilin-phosphatase Slingshot (SSH). Although cofilin is dephosphorylated in response to various extracellular stimuli, signaling pathways regulating SSH activation and cofilin dephosphorylation have remained to be elucidated. Here we show that insulin stimulates the phosphatase activity of Slingshot-1L (SSH1L) and cofilin dephosphorylation in cultured cells, in a manner dependent on phosphoinositide 3-kinase (PI3K) activity. Consistent with this, the level of Ser-3-phosphorylated cofilin is increased in PTEN (phosphatase and tensin homolog deleted in chromosome 10)-overexpressing cells and decreased in PTEN-deficient cells. Insulin induced the accumulation of SSH1L and active Akt (a downstream effector of PI3K), together with a PI3K product phosphatidylinositol 3,4,5-trisphosphate, onto membrane protrusions. Cofilin, but not Ser-3-phosphorylated cofilin, accumulated in membrane protrusions in insulin-stimulated cells, indicating that cofilin is dephosphorylated in these areas. Finally, suppression of SSH1L expression by RNA interference abolished insulin-induced cofilin dephosphorylation and the membrane protrusion. These findings suggest that SSH1L is activated downstream of PI3K and plays a critical role in insulin-induced membrane protrusion by dephosphorylating and activating cofilin. Cell migration plays a central role in a variety of physiological and pathological events, including wound healing, inflammation, immune responses, embryogenesis, organogenesis, angiogenesis, and tumor invasion and metastasis. Lamellipodial membrane protrusions are formed in the initial stage of cell movement and maintained at the leading edge of the cell throughout polarized cell migration (1Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar). Rapid turnover of actin filaments is essential for the formation and maintenance of membrane protrusions for cell migration (1Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar, 2Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (844) Google Scholar, 3Bailly M. Jones G.E. Curr. Biol. 2003; 13: R128-130Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In membrane protrusions, actin filaments are assembled and organized as a dendritic network by actions of the Arp2/3 complex, which stimulates nucleation and branching of actin filaments (1Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar). As a branched network of actin filaments grows by actin assembly at the barbed ends in the front side of membrane protrusions, it pushes the membrane forward (1Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar). On the other hand, actin filaments are rapidly disassembled at the pointed ends on the rear side of membrane protrusions, which is mediated by cofilin and its close relative, actin-depolymerizing factor (ADF) 1The abbreviations used are: ADF, actin depolymerizing factor; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; LIMK1, LIM kinase-1; MEF, mouse embryonic fibroblast; P-Akt, Ser-473-phosphorylated Akt; P-cofilin, Ser-3-phosphorylated cofilin; PHAKT, PH domain of Akt; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog deleted in chromosome 10; RNAi, RNA interference; SSH, Slingshot; SSH1L, a long isoform of Slingshot-1; WT, wild-type; CS, phosphatase-dead. (1Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar, 2Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (844) Google Scholar, 3Bailly M. Jones G.E. Curr. Biol. 2003; 13: R128-130Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Cofilin and ADF (hereafter referred to as cofilin) stimulate depolymerization and severance of actin filaments at or near the pointed ends and thereby supply actin monomers for polymerization and support rapid turnover of actin filaments (1Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar, 2Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (844) Google Scholar, 3Bailly M. Jones G.E. Curr. Biol. 2003; 13: R128-130Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The filament-severing activity of cofilin also contributes to the appearance of free barbed ends, from which actin filaments can grow to the leading edge (4Zebda N. Bernard O. Bailly M. Welti S. Lawrence D.S. Condeelis J.S. J. Cell Biol. 2000; 151: 1119-1128Crossref PubMed Scopus (170) Google Scholar). Thus, the activity of cofilin seems to play an essential role in maintaining continuous extension of the plasma membrane at the leading edge of locomoting cells (3Bailly M. Jones G.E. Curr. Biol. 2003; 13: R128-130Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 4Zebda N. Bernard O. Bailly M. Welti S. Lawrence D.S. Condeelis J.S. J. Cell Biol. 2000; 151: 1119-1128Crossref PubMed Scopus (170) Google Scholar, 5Endo M. Ohashi K. Sasaki Y. Goshima Y. Niwa R. Uemura T. Mizuno K. J. Neurosci. 2003; 23: 2527-2537Crossref PubMed Google Scholar). The activity of cofilin is negatively regulated by serine/threonine kinases, LIM kinase (LIMK) and TESK, through phosphorylation at Ser-3 of cofilin (6Yang 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, 7Arber 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, 8Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (222) Google Scholar). LIMK1 is activated in response to various extracellular stimuli, including lysophosphatidic acid, stromal cell-derived factor-1α, and insulin, through Rho family small GTPases, Rho, Rac, and Cdc42, and their downstream protein kinases, such as ROCK and PAK (6Yang 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, 9Ohashi 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, 10Nishita M. Aizawa H. Mizuno K. Mol. Cell. Biol. 2002; 22: 774-783Crossref PubMed Scopus (117) Google Scholar). The Ser-3-phosphorylated cofilin (P-cofilin) is dephosphorylated and reactivated by a family of protein phosphatases, termed Slingshot (SSH) (11Niwa 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). SSH was originally identified in Drosophila. The loss of SSH function in Drosophila leads to disorganized epidermal cell morphogenesis, including malformation of bristles, wing hairs, and ommatidia. Thus, SSH is implicated in the formation of cellular extensions by organizing the ordered assembly of actin filaments in Drosophila (11Niwa 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). In mammals, members of a SSH phosphatase family, SSH1L, SSH2L, and SSH3L, were found to have distinct specific activities, subcellular distribution, and tissue expression patterns (11Niwa 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, 12Ohta 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). Recently, we showed that the phosphatase activity of SSH1L changes during the cell division cycle in cultured cells (13Kaji 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). Previous studies revealed that cofilin is dephosphorylated in response to various extracellular stimuli (2Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (844) Google Scholar, 14Moon A. Drubin D.G. Mol. Biol. Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (227) Google Scholar, 15Meberg 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, signaling mechanisms that regulate SSH activity and cofilin dephosphorylation have remained to be elucidated. Phosphoinositide 3-kinase (PI3K) is a key mediator of intracellular signaling pathways that regulate metabolism, growth, apoptosis, actin cytoskeletal reorganization, and polarized cell migration (16Saltiel A.R. Pessin J.E. Trends Cell Biol. 2002; 12: 65-71Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 17Chung C.Y. Funamoto S. Firtel R.A. Trends Biochem. Sci. 2001; 26: 557-566Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 18Rickert P. Weiner O.D. Wang F. Bourne H.R. Servant G. Trends Cell Biol. 2000; 10: 466-473Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Activated PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to generate phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Production of PI(3,4,5)P3 is followed by activation of downstream effectors, such as Rac and a serine/threonine kinase Akt (also called protein kinase B) (16Saltiel A.R. Pessin J.E. Trends Cell Biol. 2002; 12: 65-71Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 17Chung C.Y. Funamoto S. Firtel R.A. Trends Biochem. Sci. 2001; 26: 557-566Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 18Rickert P. Weiner O.D. Wang F. Bourne H.R. Servant G. Trends Cell Biol. 2000; 10: 466-473Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). A tumor suppressor protein PTEN (phosphatase and tensin homolog deleted in chromosome 10), which is frequently mutated or deleted in various human cancers, antagonizes PI3K signaling by dephosphorylating PI(3,4,5)P3 to PI(4,5)P2 (19Maehama T. Dixon J.E. Trends Cell Biol. 1999; 9: 125-128Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). PTEN deficiency results in the accumulation of PI(3,4,5)P3 in cells and an increase in cell motility in mouse embryonic fibroblasts (MEFs) (20Higuchi M. Masuyama N. Fukui Y. Suzuki A. Gotoh Y. Curr. Biol. 2001; 11: 1958-1962Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 21Liliental J. Moon S.Y. Lesche R. Mamillapalli R. Li D. Zheng Y. Sun H. Wu H. Curr. Biol. 2000; 10: 401-404Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 22Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2120) Google Scholar). Activation of Rac downstream of PI3K has been shown to be essential for the formation of membrane protrusions (23Srinivasan S. Wang F. Glavas S. Ott A. Hofmann F. Aktories K. Kalman D. Bourne H.R. J. Cell Biol. 2003; 160: 375-385Crossref PubMed Scopus (364) Google Scholar). Although these observations suggest the critical roles of PI3K in membrane protrusion and cell migration, it has remained to be determined how PI3K regulates actin filament dynamics, membrane protrusion formation, and polarized cell migration. We now provide evidence that PI3K regulates cofilin dephosphorylation through activation of SSH1L and that PI3K-mediated SSH1L activation is essential for insulin-induced membrane protrusion. Our results suggest a novel signaling mechanism by which PI3K controls actin filament dynamics and membrane protrusion. Materials—Insulin, wortmannin, and monoclonal antibodies against Akt were purchased from Sigma. Monoclonal and rabbit polyclonal antibodies against Ser-473-phosphorylated Akt (P-Akt) and rabbit polyclonal antibody against PTEN were from Cell Signaling Technology (Beverly, MA). Monoclonal antibody against Myc (9E10) was purchased form Roche Diagnostics. Rabbit polyclonal antibodies to P-cofilin, cofilin, LIMK1, and SSH1L were prepared as described (8Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (222) Google Scholar, 9Ohashi 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, 13Kaji 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, 24Okano I. Hiraoka J. Otera H. Nunoue K. Ohashi K. Iwashita S. Hirai M. Mizuno K. J. Biol. Chem. 1995; 270: 31321-31330Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Plasmids—Plasmids coding for Myc-tagged SSH1L and PTEN (wild-type (WT) and phosphatase-dead (CS)) were constructed as described (11Niwa 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, 22Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2120) Google Scholar). The plasmid for cyan fluorescent protein (CFP)-tagged SSH1L (CFP-SSH1L) was constructed by subcloning SSH1L cDNA into pECFP-C1 (Clontech). Plasmid for yellow fluorescent protein (YFP)-tagged PH domain of Akt (PHAKT) was constructed by subcloning cDNA for the PH domain of rat Akt1 (25Konishi H. Matsuzaki H. Tanaka M. Ono Y. Tokunaga C. Kuroda S. Kikkawa U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7639-7643Crossref PubMed Scopus (189) Google Scholar) into pEYFP-C1 (Clontech). The pSUPER vector for RNA interference (RNAi) was kindly provided by R. Agami (The Netherlands Cancer Institute) (26Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar). An SSH1L RNAi plasmid (pSUPER-SSH1L) that targets human SSH1L mRNA sequence (5′-TCGTCACCCAAGAAAGATA-3′) was constructed as described (26Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar). Cell Culture, Transfection, and RNA Interference—293T, MCF-7, and PTEN+/- and PTEN-/- MEFs (22Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2120) Google Scholar) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cells were transfected with expression plasmids by using the FuGENE6 Transfection Reagent (Roche Diagnostics) according to the manufacturer's instructions. For RNAi, pSUPER-SSH1L was electroporated into MCF-7 cells; about 2 × 106 cells were mixed with plasmid (20 μg) in 400 μl of medium (Dulbecco's modified Eagle's medium containing 20% fetal calf serum and 25 mm HEPES, pH 7.4) and electroporated at 250 V and 975 microfarads, using Gene Pulser II (Bio-Rad). Cells were used for assays after a 60-h of culture. Phosphatase Assay—Cofilin-His6 expressed in Vero cells was purified and used as a substrate for in vitro phosphatase reaction. 293T cells were lysed in phosphatase buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 5% glycerol, 1 mm dithiothreitol, 10 μg/ml leupeptin). SSH1L was immunoprecipitated with anti-SSH1L antibody and incubated with purified cofilin-His6 for2hat30 °C. Reaction mixtures were analyzed by immunoblotting using antibodies specific to cofilin, P-cofilin, and SSH1L. Kinase Assay—MCF-7 cells were lysed in lysis/kinase buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 0.5% Nonidet P-40, 5% glycerol, 1 mm MgCl2, 1 mm MnCl2, 20 mm NaF, 1 mm Na3VO4, 1 mm dithiothreitol, 0.25 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin). LIMK1 was immunoprecipitated with anti-LIMK1 antibody and subjected to in vitro kinase reaction as described (9Ohashi 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). Cell Staining—MCF-7 cells were fixed with 4% formaldehyde. For staining with antibodies, fixed cells were incubated with 100% methanol. After blocking with 5 mg/ml bovine serum albumin, cells were incubated with mouse monoclonal antibody to P-Akt and rabbit polyclonal antibody to either cofilin or P-cofilin. Rhodamine-phalloidin and 4′,6-diamidino-2-phenylindole were used to stain F-actin and DNA, respectively. Fluorescent images were obtained using a Zeiss LSM510 laser scanning confocal microscope or a Leica DMLB fluorescence microscope. Insulin Induces Cofilin Dephosphorylation and SSH1L Activation—Insulin stimulates actin cytoskeletal reorganization and induces changes in cell morphology and motility in a variety of cell types (16Saltiel A.R. Pessin J.E. Trends Cell Biol. 2002; 12: 65-71Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 27Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar, 28Tobe K. Asai S. Matuoka K. Yamamoto T. Chida K. Kaburagi Y. Akanuma Y. Kuroki T. Takenawa T. Kimura S. Nagai R. Kadowaki T. Genes Cells. 2003; 8: 29-40Crossref PubMed Scopus (15) Google Scholar). To investigate signaling pathways that regulate cofilin activity, we first determined whether insulin stimulation would alter the level of cofilin phosphorylation in cultured cells. Immunoblot analysis with an antibody specific to P-cofilin revealed that exposure of 293T cells with insulin significantly decreased the level of P-cofilin in a time-dependent manner without affecting the total cofilin level (Fig. 1A), a finding which indicates that insulin induces cofilin dephosphorylation in 293T cells. Since insulin was shown to lead to activation of LIMK1 (6Yang 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) (see also Fig. 5B), this finding raises the possibility that insulin induces activation of cofilin-phosphatases, such as SSH1L, over the activation of LIMK1 to decrease the total P-cofilin level. To test this possibility, we analyzed changes in phosphatase activity of endogenous SSH1L after insulin stimulation. SSH1L was immunoprecipitated with anti-SSH1L antibody and incubated with recombinant cofilin-His6 substrates that contain P-cofilin. As shown in Fig. 1B, insulin exposure induced activation of SSH1L in a time course similar to that of cofilin dephosphorylation. These observations suggest that insulin induces cofilin dephosphorylation through activation of SSH1L.Fig. 5RNAi knockdown of SSH1L inhibits insulin-induced cofilin dephosphorylation. A, suppression of SSH1L expression by RNAi. MCF-7 cells were electroporated with control or SSH1L RNAi plasmids, cultured for 44 h, and serum-starved for 16 h. Expression of endogenous SSH1L and LIMK1 was analyzed by immunoblotting with anti-SSH1L and anti-LIMK1 antibodies. As shown in B, SSH1L RNAi abolishes insulin-induced cofilin dephosphorylation. MCF-7 cells transfected with RNAi plasmids were serum-starved and stimulated with insulin for the indicated periods, and cell lysates were immunoblotted for P-cofilin, cofilin, P-Akt, and Akt, as indicated. For kinase activity of LIMK1, cell lysates were immunoprecipitated with anti-LIMK1 antibody and subjected to in vitro kinase reaction, using cofilin-His6 as a substrate. Reaction mixtures were run on SDS-PAGE and analyzed by autoradiography (32P-cofilin) and Amido Black staining (cofilin).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Insulin-induced Cofilin Dephosphorylation and SSH1L Activation Depend on PI3K Activity—PI3K is implicated in many cellular responses, including metabolism, growth, apoptosis, actin reorganization, and polarized cell migration (16Saltiel A.R. Pessin J.E. Trends Cell Biol. 2002; 12: 65-71Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 17Chung C.Y. Funamoto S. Firtel R.A. Trends Biochem. Sci. 2001; 26: 557-566Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 18Rickert P. Weiner O.D. Wang F. Bourne H.R. Servant G. Trends Cell Biol. 2000; 10: 466-473Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). This kinase plays a major role in insulin signaling pathways, but the role of PI3K in insulin-induced actin reorganization has remained unclear (16Saltiel A.R. Pessin J.E. Trends Cell Biol. 2002; 12: 65-71Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 28Tobe K. Asai S. Matuoka K. Yamamoto T. Chida K. Kaburagi Y. Akanuma Y. Kuroki T. Takenawa T. Kimura S. Nagai R. Kadowaki T. Genes Cells. 2003; 8: 29-40Crossref PubMed Scopus (15) Google Scholar). When we examined effects of wortmannin and LY294002, specific inhibitors for PI3K, on insulin-induced cofilin dephosphorylation and SSH1L activation, we found that preincubation of 293T cells with wortmannin or LY294002 inhibited both (Fig. 1, C and D, top panel). By monitoring the level of P-Akt, an active form of Akt that is produced following PI3K activation (29Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar), we confirmed the efficient inhibition of PI3K activity by these inhibitors (Fig. 1C, third panel). These results strongly suggest that PI3K mediates insulin-induced SSH1L activation and cofilin dephosphorylation. In addition, the level of P-cofilin was decreased by ectopic expression of SSH1L, and it was reverted to the control level by treatment of cells with wortmannin for 1 h (Fig. 1E), which suggests that PI3K activity is critical for SSH1L to elicit cofilin-dephosphorylating activity in cultured cells. PTEN Negatively Regulates Cofilin Dephosphorylation—PTEN antagonizes PI3K signaling by dephosphorylating PI(3,4,5)P3 (19Maehama T. Dixon J.E. Trends Cell Biol. 1999; 9: 125-128Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). To further examine the involvement of PI3K and its product PI(3,4,5)P3 in the regulation of cofilin dephosphorylation, we overexpressed either WT or CS PTEN in 293T cells and analyzed the P-cofilin level. Expression of PTEN(WT) led to a significant increase in the P-cofilin level, whereas PTEN(CS) slightly reduced it (Fig. 2A). Co-expression of PTEN(WT) with SSH1L suppressed the cofilin dephosphorylation induced by SSH1L, whereas PTEN(CS) did not do so (Fig. 2B), which further supports the notion that cofilin-phosphatase activity of SSH1L depends on the level of PI(3,4,5)P3. We next compared cellular P-cofilin levels in PTEN+/- and PTEN-/- MEFs (22Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2120) Google Scholar). As Stambolic et al. reported (22Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2120) Google Scholar), the level of P-Akt was elevated in PTEN-/- MEFs, as compared with that in PTEN+/- MEFs (Fig. 2C, third panel). Cofilin and ADF were phosphorylated to a lesser extent in PTEN-/- MEF cells (Fig. 2C, top panel). These results suggest that PTEN negatively regulates cofilin dephosphorylation, probably by decreasing the cellular content of PI(3,4,5)P3. Contrarily, PI3K and its product PI(3,4,5)P3 seem to play a role in promoting cofilin dephosphorylation and reactivation. Accumulation of SSH1L and PI(3,4,5)P3 in Insulin-induced Membrane Protrusions—To examine the subcellular localization of SSH1L and PI(3,4,5)P3 before and after insulin stimulation, we used CFP-tagged SSH1L and YFP-tagged PHAKT (a PH domain of Akt), which specifically binds to PI(3,4,5)P3 and was utilized to assign the spatial distribution of PI(3,4,5)P3 (30Servant G. Weiner O.D. Herzmark P. Balla T. Sedat J.W. Bourne H.R. Science. 2000; 287: 1037-1040Crossref PubMed Scopus (737) Google Scholar). To avoid the inhibitory effect on downstream signaling of PI3K by high expression of PHAKT (31Wang Q. Liu L. Pei L. Ju W. Ahmadian G. Lu J. Wang Y. Liu F. Wang Y.T. Neuron. 2003; 38: 915-928Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), MCF-7 cells were transfected with expression plasmids for CFP-SSH1L and YFP-PHAKT with a molar ratio of 5:1. In the absence of insulin, cells had a flat shape, and both CFP-SSH1L and YFP-PHAKT were diffusely localized in the cytoplasm and cell periphery (Fig. 3A, upper panels). In contrast, after exposure to insulin, the cells altered their shapes with several F-actin-rich membrane protrusions (Fig. 3A, lower panels). In insulin-stimulated cells, both CFP-SSH1L and YFP-PHAKT co-localized with F-actin and accumulated together onto membrane protrusions (Fig. 3A, lower panels). These observations suggest that insulin stimulation leads to accumulation of PI(3,4,5)P3 and SSH1L onto membrane protrusions. Inhibition of PI3K activity by wortmannin almost completely blocked insulin-induced cell shape changes and accumulation of PI(3,4,5)P3 and SSH1L in membrane protrusions (Fig. 3B). These results suggest that accumulation of SSH1L to membrane protrusions depends on PI3K activity and that local accumulation of PI(3,4,5)P3 is critical for recruitment of SSH1L to membrane protrusions. Accumulation of Cofilin, but Not P-cofilin, in Insulin-induced Membrane Protrusions—Accumulation of CFP-SSH1L in membrane protrusions of insulin-stimulated cells suggests that cofilin dephosphorylation preferentially occurs at the protrusions after insulin stimulation. We examined the localization of cofilin and P-cofilin, together with P-Akt (as a marker for activation of PI3K-Akt pathway), in MCF-7 cells before and after insulin stimulation. In non-stimulated cells, both cofilin and P-cofilin distributed uniformly throughout the cell, and the P-Akt signal was barely detectable (Fig. 4, A and B, upper panels). Interestingly, insulin stimulation induced a significant accumulation of cofilin and P-Akt at membrane protrusions (Fig. 4A, lower panels). In contrast, P-cofilin was diffusely distributed in the cytoplasm and was not concentrated in the regions of membrane protrusions where P-Akt was concentrated; therefore, P-cofilin signals poorly overlapped with P-Akt signals (Fig. 4B, lower panels). Thus, non-phosphorylated (active) cofilin is the major component of cofilin signals that accumulated in membrane protrusions. Because SSH1L accumulates in insulin-induced membrane protrusions, it probably functions to stimulate cofilin dephosphorylation. Similar to our observations, Dawe et al. (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) recently showed that cofilin exists at the leading edge of migrating fibroblasts but P-cofilin was depleted from it. Accordingly, enhancement of cofilin dephosphorylation by SSH1L may generally occur at membrane protrusions where rapid actin turnover is required. SSH1L Is Essential for Insulin-induced Cofilin Dephosphorylation and Morphological Changes—To further examine the cellular function of SSH1L in insulin-induced cofilin dephosphorylation and cell morphological changes, SSH1L expression in MCF-7 cells was suppressed by RNAi. We transfected by electroporation control or SSH1L RNAi plasmids (26Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar) that direct the synthesis of short interfering RNAs, into MCF-7 cells. Transfection efficiency exceeded 90%. Immunoblot analysis revealed that transfection of SSH1L RNAi plasmids efficiently suppressed expression of endogenous SSH1L protein but had no apparent effect on LIMK1 expression (Fig. 5A). Suppression of SSH1L expression led to an increase in the basal level of P-cofilin in non-stimulated cells (Fig. 5B, top panel, 0 min). In cells transfected with a control RNAi vector, insulin stimulation induced rapid dephosphorylation of cofilin (Fig. 5B, top panel). In contrast, in cells transfected with SSH1L RNAi plasmids, the levels of P-cofilin did not change (or slightly increased) after insulin treatment for 5-30 min (Fig. 5B, top panel), which suggests that SSH1L plays an essential role in insulin-induced cofilin dephosphorylation. In parallel experiments, RNAi of SSH1L did not affect the insulin-induced phosphorylation of Akt (Fig. 5B, third panel) and activation of LIMK1 (Fig. 5B, fifth panel). We then examined the effects of SSH1L RNAi on insulin-induced actin reorganization and cell morphological changes in MCF-7 cells. SSH1L RNAi had no apparent effect in the absence of insulin (Fig. 6A, upper panels), but it remarkably suppressed insulin-induced cell shape changes, including cell dissemination and membrane protrusion formation (Fig. 6A, lower panels). Quantitative analyses of the percentages of cells with extended membrane protrusions before and after insulin treatment further indicate that SSH1L RNAi suppressed the membrane protrusion formation (Fig. 6B). Pretreatment with wortmannin also inhibited insulin-induced morphological changes (Fig. 6A). These results suggest that SSH1L as well as PI3K play a critical role in insulin-induced cell shape alterations. On the other hand, phalloidin staining revealed that SSH1L RNAi did not suppress insulin-induced F-actin assembly, whereas wortmannin did do so (Fig. 6A, arrows). Thus, PI3K, but not SSH1L, seems to be involved in insulin-induced actin filament assembly. Cofilin activity is reversibly regulated by phosphorylation and dephosphorylation at Ser-3. Phosphorylation of cofilin by LIMK or TESK hinders the interaction between cofilin and actin, thereby inhibiting actin-depolymerizing and -severing activity of cofilin. Phosphorylated cofilin is dephosphorylated and reactivated by SSH (11Niwa 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). Although LIMK1 is known to be activated in response to various extracellular stimuli, including lysophosphatidic acid, stromal cell-derived factor-1α, and insulin, through Rho family small GTPases (6Yang 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, 9Ohashi 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, 10Nishita M. Aizawa H. Mizuno K. Mol. Cell. Biol. 2002; 22: 774-783Crossref PubMed Scopus (117) Google Scholar), it remained to be determined whether the activity of SSH is regulated in response to extracellular stimuli, and if so, what signaling components are involved in such regulatory mechanisms. In this study, we demonstrated that the phosphatase activity of SSH1L is stimulated by insulin. Using specific inhibitors for PI3K, we also provided evidence that PI3K mediates insulin-induced SSH1L activation and cofilin dephosphorylation. Consistent with these results, loss of PTEN gene in MEFs led to reduction of P-cofilin levels, and overexpression of PTEN increased P-cofilin levels. Since PTEN antagonizes PI3K activity by dephosphorylating PI(3,4,5)P3, the intracellular level of PI(3,4,5)P3 seems to play a critical role in determining the level of cofilin phosphorylation and SSH1L activity. Since PTEN-/- MEFs migrate faster than PTEN+/- or wild-type MEFs (20Higuchi M. Masuyama N. Fukui Y. Suzuki A. Gotoh Y. Curr. Biol. 2001; 11: 1958-1962Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 21Liliental J. Moon S.Y. Lesche R. Mamillapalli R. Li D. Zheng Y. Sun H. Wu H. Curr. Biol. 2000; 10: 401-404Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), an intriguing possibility is that an increase in the content of the non-phosphorylated (active) form of cofilin in PTEN-/- MEFs contributes, at least in part, to their higher migrating activity. It has been unknown whether other SSH family members (i.e. SSH2L and SSH3L) are also activated in response to insulin stimulation. However, because SSH1L RNAi almost completely suppressed the insulin-induced cofilin dephosphorylation in MCF-7 cells, SSH1L seems to be a major cofilin phosphatase that functions in response to insulin in MCF-7 cells. Actin filament assembly occurs as an initial step in membrane protrusion. Since SSH1L RNAi did not block insulin-induced actin filament assembly, SSH1L might not be essential for this step of membrane protrusion. In the later stages of membrane protrusion, rapid turnover of actin filaments is critical, composed of continuous assembly at the barbed ends and concomitant disassembly at the pointed ends. As cofilin plays an essential role in actin filament turnover by depolymerizing and severing actin filaments (1Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar, 2Bamburg J.R. Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (844) Google Scholar, 3Bailly M. Jones G.E. Curr. Biol. 2003; 13: R128-130Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), our results suggest that SSH1L accumulating in membrane protrusions plays a critical role in the later stages of membrane protrusion by dephosphorylating and reactivating cofilin. In support of this idea, overexpression of active LIMK1 that causes aberrant phosphorylation and inactivation of cofilin suppressed membrane protrusion and cell motility (4Zebda N. Bernard O. Bailly M. Welti S. Lawrence D.S. Condeelis J.S. J. Cell Biol. 2000; 151: 1119-1128Crossref PubMed Scopus (170) Google Scholar, 5Endo M. Ohashi K. Sasaki Y. Goshima Y. Niwa R. Uemura T. Mizuno K. J. Neurosci. 2003; 23: 2527-2537Crossref PubMed Google Scholar). On the other hand, insulin activates LIMK1, and insulin-induced membrane ruffling is inhibited by kinase-dead LIMK1 (6Yang 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). Thus, spatially and temporarily coordinated activation of both LIMK1 and SSH1L seems to be important for insulin-induced membrane protrusion. Well controlled activation of LIMK1 induced by insulin may play a role in the initial step of membrane protrusion by stabilizing actin filaments. Otherwise, LIMK1 may contribute to rapid actin turnover in cooperation with SSH1L by accelerating the recycling of cofilin and actin by releasing free actin and cofilin from an actin-cofilin complex, which is generated from the pointed end of actin filaments by the depolymerizing action of cofilin (33Rosenblatt J. Mitchison T.J. Nature. 1998; 393: 739-740Crossref PubMed Scopus (33) Google Scholar). Our results suggest that SSH1L is activated and recruited to membrane protrusions downstream of PI3K activity and that it plays a critical role in insulin-induced membrane protrusion and cell shape changes. Although this study focused on insulin action, we have data demonstrating that cell stimulation with other growth factors and chemokines that stimulate cell motility also induces SSH1L activation and cofilin dephosphorylation through PI3K activation. Given the essential roles of cofilin and PI3K in actin cytoskeletal reorganization and polarized cell movement (3Bailly M. Jones G.E. Curr. Biol. 2003; 13: R128-130Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 34Weiner O.D. Curr. Opin. Cell Biol. 2002; 14: 196-202Crossref PubMed Scopus (212) Google Scholar), further studies on signaling pathways linking PI3K activity to SSH1L activation will be important to elucidate mechanisms governing how cells establish and maintain actin filament dynamics at the leading edge of migrating cells for directed cell movement. We thank K. Goto for technical assistance, Dr. R. Agami for the pSUPER plasmid, Dr. U. Kikkawa for Akt plasmids, and M. Ohara for helpful comments." @default.
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W2028497798 date "2004-02-01" @default.
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W2028497798 title "Phosphoinositide 3-Kinase-mediated Activation of Cofilin Phosphatase Slingshot and Its Role for Insulin-induced Membrane Protrusion" @default.
W2028497798 cites W1484094963 @default.
W2028497798 cites W1640813462 @default.
W2028497798 cites W1978002465 @default.
W2028497798 cites W1998038367 @default.
W2028497798 cites W1999920891 @default.
W2028497798 cites W2003736664 @default.
W2028497798 cites W2007660746 @default.
W2028497798 cites W2009718333 @default.
W2028497798 cites W2009756576 @default.
W2028497798 cites W2012785062 @default.
W2028497798 cites W2020891498 @default.
W2028497798 cites W2020931266 @default.
W2028497798 cites W2038783205 @default.
W2028497798 cites W2039470997 @default.
W2028497798 cites W2041607472 @default.
W2028497798 cites W2050002753 @default.
W2028497798 cites W2050498522 @default.
W2028497798 cites W2064523806 @default.
W2028497798 cites W2064879077 @default.
W2028497798 cites W2067246434 @default.
W2028497798 cites W2072706075 @default.
W2028497798 cites W2082971375 @default.
W2028497798 cites W2090206221 @default.
W2028497798 cites W2099322968 @default.
W2028497798 cites W2105286896 @default.
W2028497798 cites W2116052541 @default.
W2028497798 cites W2126496496 @default.
W2028497798 cites W2126546276 @default.
W2028497798 cites W2141617119 @default.
W2028497798 cites W2143073532 @default.
W2028497798 cites W2150141558 @default.
W2028497798 cites W2151519283 @default.
W2028497798 cites W2160910785 @default.
W2028497798 cites W2770415951 @default.
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