Interaction of Filamin A with the Insulin Receptor Alters Insulin-dependent Activation of the Mitogen-activated Protein Kinase Pathway
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m301003200
ISSN1083-351X
AutoresHua‐Jun He, Sutapa Kole, Yong-Kook Kwon, Michael T. Crow, Michel Bernier,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe biological actions of insulin are associated with a rapid reorganization of the actin cytoskeleton within cells in culture. Even though this event requires the participation of actin-binding proteins, the effect of filamin A (FLNa) on insulin-mediated signaling events is still unknown. We report here that human melanoma M2 cells lacking FLNa expression exhibited normal insulin receptor (IR) signaling, whereas FLNa-expressing A7 cells were unable to elicit insulin-dependent Shc tyrosine phosphorylation and p42/44 MAPK activation despite no significant defect in IR-stimulated phosphorylation of insulin receptor substrate-1 or activation of the phosphatidylinositol 3-kinase/AKT cascade. Insulin-dependent translocation of Shc, SOS1, and MAPK to lipid raft microdomains was markedly attenuated by FLNa expression. Coimmunoprecipitation experiments and in vitro binding assays demonstrated that FLNa binds constitutively to IR and that neither insulin nor depolymerization of actin by cytochalasin D affected this interaction. The colocalization of endogenous FLNa with IR was detected at the surface of HepG2 cells. Ectopic expression of a C-terminal fragment of FLNa (FLNaCT) in HepG2 cells blocked the endogenous IR-FLNa interaction and potentiated insulin-stimulated MAPK phosphorylation and transactivation of Elk-1 compared with vector-transfected cells. Expression of FLNaCT had no major effect on insulin-induced phosphorylation of the IR, insulin receptor substrate-1, or AKT, but it elicited changes in actin cytoskeletal structure and ruffle formation in HepG2 cells. Taken together, these results indicate that FLNa interacts constitutively with the IR to exert an inhibitory tone along the MAPK activation pathway. The biological actions of insulin are associated with a rapid reorganization of the actin cytoskeleton within cells in culture. Even though this event requires the participation of actin-binding proteins, the effect of filamin A (FLNa) on insulin-mediated signaling events is still unknown. We report here that human melanoma M2 cells lacking FLNa expression exhibited normal insulin receptor (IR) signaling, whereas FLNa-expressing A7 cells were unable to elicit insulin-dependent Shc tyrosine phosphorylation and p42/44 MAPK activation despite no significant defect in IR-stimulated phosphorylation of insulin receptor substrate-1 or activation of the phosphatidylinositol 3-kinase/AKT cascade. Insulin-dependent translocation of Shc, SOS1, and MAPK to lipid raft microdomains was markedly attenuated by FLNa expression. Coimmunoprecipitation experiments and in vitro binding assays demonstrated that FLNa binds constitutively to IR and that neither insulin nor depolymerization of actin by cytochalasin D affected this interaction. The colocalization of endogenous FLNa with IR was detected at the surface of HepG2 cells. Ectopic expression of a C-terminal fragment of FLNa (FLNaCT) in HepG2 cells blocked the endogenous IR-FLNa interaction and potentiated insulin-stimulated MAPK phosphorylation and transactivation of Elk-1 compared with vector-transfected cells. Expression of FLNaCT had no major effect on insulin-induced phosphorylation of the IR, insulin receptor substrate-1, or AKT, but it elicited changes in actin cytoskeletal structure and ruffle formation in HepG2 cells. Taken together, these results indicate that FLNa interacts constitutively with the IR to exert an inhibitory tone along the MAPK activation pathway. Insulin is a pleiotropic hormone with multiple integrated metabolic and mitogenic signaling pathways. Upon binding of insulin, the cell surface insulin receptor (IR) 1The abbreviations used are: IR, insulin receptor; FLNa, filamin A; MAPK, mitogen-activated protein kinase; IRS-1, insulin receptor substrate 1; SH2, Src homology 2 domain; PI3-kinase, phosphatidylinositol 3-kinase; PBS, phosphate-buffered saline; MES, 4-morpholinoethanesulfonic acid; MEM, minimum essential medium; EGF, epidermal growth factor; FBS, fetal bovine serum; IGF-1, insulin-like growth factor 1; GST, glutathione S-transferase; HA, hemagglutinin; ERK, extracellular signal-regulated kinase. undergoes autophosphorylation on several tyrosine residues located in the cytoplasmic portion of the β-subunit, with subsequent increase in its intrinsic tyrosine kinase activity. A number of adaptor proteins, including insulin receptor substrate (IRS) 1/2, the Src and collagen homologous (Shc) molecules, Cbl, Gab1, p60Dok, and APS, are recruited to the activated IR through their Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains and become tyrosine-phosphorylated, thus allowing formation of signaling competent complexes subjacent to the inner wall of the plasma membrane (1Saltiel A.R. Pessin J.E. Trends Cell Biol. 2002; 12: 65-71Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). Specificity in signaling is achieved through differences in the ability of IR to interact with these adaptor molecules. The submembranous actin microfilament network links various signaling proteins (e.g. phosphatidylinositol (PI) 3-kinase and Rho family of proteins) that play an important role in membrane trafficking, cellular integrity, and homeostasis (2Okkenhaug K. Vanhaesebroeck B. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/OC_sigtrans;2001/65/pe1)PubMed Google Scholar, 3Takenawa T. Itoh T. Biochim. Biophys. Acta. 2001; 1533: 190-206Crossref PubMed Scopus (245) Google Scholar, 4Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). Incidentally, insulin is known to induce rapid dynamic reorganization of the actin cytoskeleton to generate the forces necessary for plasma membrane ruffling formation and a host of other cellular processes, including proper insertion of insulin-regulatable glucose transporter 4 in the cell surface (5Khayat Z.A. Tong P. Yaworsky K. Bloch R.J. Klip A. J. Cell Sci. 2000; 113: 279-290Crossref PubMed Google Scholar). The stabilization of actin network at the periphery of the cytoplasm and its attachment to cellular membranes are orchestrated by actin-binding proteins. The filamin family of actin-binding proteins bind to actin filaments and to a number of macromolecules (reviewed by Stossel et al. (6Stossel T.P. Condeelis J. Cooley L. Hartwig J.H. Noegel A. Schleicher M. Shapiro S.S. Nat. Rev. Mol. Cell Biol. 2001; 2: 138-145Crossref PubMed Scopus (827) Google Scholar)), notably small GTPases (7Ohta Y. Suzuki N. Nakamura S. Hartwig J.H. Stossel T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2122-2128Crossref PubMed Scopus (373) Google Scholar) and p21-activated kinase 1 (Pak1) (8Vadlamudi R.K. Li F. Adam L. Nguyen D. Ohta Y. Stossel T.P. Kumar R. Nat. Cell Biol. 2002; 4: 681-690Crossref PubMed Scopus (263) Google Scholar). Filamins are rod-shaped proteins of ∼280 kDa that contain an N-terminal actin-binding domain followed by 24 repeats each of 96 amino acids. Repeat 24 contains the dimerization domain of filamin. Significantly, most of the interactions between filamin and its binding partners occur through the C-terminal end of filamins, thus allowing the interwebbing of actin scaffolds with membrane-bound proteins (6Stossel T.P. Condeelis J. Cooley L. Hartwig J.H. Noegel A. Schleicher M. Shapiro S.S. Nat. Rev. Mol. Cell Biol. 2001; 2: 138-145Crossref PubMed Scopus (827) Google Scholar). Several proteins involved in signal transduction events are partitioned in lipid rafts, a process that allows proper compartmentalization and spatial/temporal organization of functionally competent signaling complexes (9Zajchowski L.D. Robbins S.M. Eur. J. Biochem. 2002; 269: 737-752Crossref PubMed Scopus (214) Google Scholar). The IR segregates to glycolipid-enriched raft domains of the plasma membrane in a variety of cell types (10Gustavsson J. Parpal S. Karlsson M. Ramsing C. Thorn H. Borg M. Lindroth M. Peterson K.H. Magnusson K.E. Stralfors P. FASEB J. 1999; 13: 1961-1971Crossref PubMed Scopus (316) Google Scholar, 11Vainio S. Heino S. Mansson J.E. Fredman P. Kuismanen E. Vaarala O. Ikonen E. EMBO Rep. 2002; 3: 95-100Crossref PubMed Scopus (143) Google Scholar). Of interest, inducible association of signaling proteins with lipid rafts has been shown to depend on the actin cytoskeleton through a mechanism involving raft coalescence (12Rodgers W. Zavzavadjian J. Exp. Cell Res. 2001; 267: 173-183Crossref PubMed Scopus (59) Google Scholar, 13Valensin S. Paccani S.R. Ulivieri C. Mercati D. Pacini S. Patrussi L. Hirst T. Lupetti P. Baldari C.T. Eur. J. Immunol. 2002; 32: 435-446Crossref PubMed Scopus (78) Google Scholar). Therefore, colocalization of filamin and resident raft proteins, including the scaffolding protein caveolin-1 (14Stahlhut M. van Deurs B. Mol. Biol. Cell. 2000; 11: 325-337Crossref PubMed Scopus (264) Google Scholar), is likely to be of physiological importance in the clustering of lipid rafts and organization of multiple signaling pathways by the actin cytoskeleton. Until now, however, little is known about a role that filamins would play in the transmission of the diverse effects of insulin. In this study, we have investigated the relative contribution of filamin A (FLNa) to the regulation of insulin signaling in human melanoma cell lines (M2 cells) that have spontaneously lost expression of FLNa, and a subline with stable expression of recombinant FLNa (A7 cells) (15Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers H.R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (498) Google Scholar). By using this cell model, we have found that FLNa expression attenuated insulin mitogenic signaling by selectively inhibiting the recruitment and tyrosine phosphorylation of Shc and subsequent activation of p42/44 MAPK. Of interest, neither early steps in insulin signaling (e.g. IR and IRS-1 tyrosine phosphorylation) nor the activation of the IRS-1/PI3-kinase/AKT pathway were affected by FLNa. MAPKs transduce a mitogenic signal by phosphorylating transcription factors such as Elk-1, which leads to regulation of critical genes (16Sharrocks A.D. Nat. Rev. Mol. Cell. Biol. 2001; 2: 827-837Crossref PubMed Scopus (819) Google Scholar). Our results have indicated also that FLNa binds directly to IR and that ectopic expression of a C-terminal fragment of FLNa disrupts constitutive IR-FLNa interaction in HepG2 cells, thereby inducing a marked increase in insulin-stimulated MAPK phosphorylation and Elk-1 transactivation. These results indicate that FLNa has a negative role in MAPK-mediated Elk-1 transcriptional activation in response to insulin, in part, by interacting directly with the IR. Materials—The anti-human IR antibodies for immunoprecipitation (clones 29B4 and CII 25.3) were purchased from Calbiochem. The anti-IR β-subunit antibody as well as horseradish peroxidase-linked phosphotyrosine (clone RC20) antibody for Western blot were from Transduction Laboratories. The anti-IR β-subunit antibody (06-492) for immunofluorescence studies, and the anti-Shc, phosphotyrosine, SOS1, IRS-1, p85 subunit of PI3-kinase, and p42/44 MAP kinase (ERK1 and 2) antibodies were from Upstate Biotechnology. Inc. The monoclonal anti-FLNa antibody for immunoprecipitation and immunofluorescence studies was purchased from Chemicon International, and that used for Western blot analysis was from Research Diagnostics Inc. The anti-IR α-subunit and c-Src antibodies were from Santa Cruz Biotechnology. The phospho-ERK and phospho-AKT (Ser(P)-473) antibodies were purchased from Promega and Cell Signaling Technology, respectively. The influenza virus hemagglutinin (HA) epitope antibodies for Western blot and immunofluorescence studies were from BD Biosciences Clontech and Covance, respectively. The Alexa Fluor secondary antibodies, Alexa Fluor-conjugated phalloidins, and Topro-3 were from Molecular Probes. The recombinant human insulin and EGF were from Calbiochem and Upstate Biotechnology. Inc., respectively. FuGENE 6 and LipofectAMINE 2000 were from Roche Applied Science and Invitrogen, respectively. Recombinant human insulin-like growth factor 1 (IGF-1), cytochalasin D, 2-mercaptoethanol, sodium orthovanadate, and Me2SO were from Sigma. The commercial sources for electrophoresis reagents, culture media, sera, films, horseradish peroxidase-linked secondary antibodies, and the enhanced chemiluminescence detection system for immunoblot detection have been described previously (17Garant M.J. Maksimova E. Montrose-Rafizadeh C. Lee-Kwon W. Kole S. Bernier M. Biochemistry. 2000; 39: 7178-7187Crossref PubMed Scopus (4) Google Scholar). Plasmid Construction—cDNA encoding the FLNa C-terminal fragment (amino acids 2357–2647) was amplified by PCR using full-length human filamin A cDNA (kindly provided by Dr. Yasutaka Ohta (Harvard Medical School, Boston)) as the template. The following primer pairs were used: forward primer, 5′-TAGGATCCATGGGCTATCCATATGATGTTCCAGATTATGCTCTGAACGGGGCCAAG-3′; reverse primer, 5′-CGACTAGTTCAGGGCACCACAAC-3′. The underlined nucleotides indicate the KpnI and SpeI sites in the forward and reverse primers, respectively, and the italic nucleotides indicate the HA epitope. The amplified product was digested and inserted into the KpnI/XbaI sites of pcDNA3.1 (Invitrogen). The integrity of the HA-FLNaCT construct was verified by automated sequencing. A cDNA fragment corresponding to Arg941–Ser1343 of the human IR was generated by PCR amplification to contain BamHI and EcoRI restriction sites using pCMVHIR as the template (18Wilden P.A. Kahn C.R. Siddle K. White M.F. J. Biol. Chem. 1992; 267: 16660-16668Abstract Full Text PDF PubMed Google Scholar). The resulting 1206-bp BamHI-EcoRI cDNA fragment was inserted into pGEX-4T-1 vector (Amersham Biosciences). GST and GST-IR fusion protein were expressed in BL21, induced by 0.5 mm isopropylthio-β-d-galactopyranoside, and purified by affinity chromatography with glutathione-Sepharose (Amersham Biosciences) according to the manufacturer's protocols. The resulting eluates were concentrated by ultrafiltration and stored at -70 °C. Translation and product size were verified by analyzing an aliquot of the samples by SDS-PAGE and Colloidal blue staining of the gel, as well as by immunoblot analysis. The integrity of the GST-IR construct was verified by automated sequencing. Cell Treatment—M2 and A7 cells were cultured in minimum essential medium (MEM) supplemented with 10 mm Hepes, pH 7.4, 0.25% sodium bicarbonate, 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 8% newborn calf serum, and 2% fetal calf serum and maintained in a humidified atmosphere of 5% CO2 in air at 37 °C. Before treatment, cells were serum-starved for 18 h in MEM supplemented with 0.1% FBS, washed with phosphate-buffered saline (PBS), and then incubated in Krebs-Ringer phosphate (KRP) buffer. Cells were treated in the absence or the presence of 200 μm orthovanadate for 30 min followed by the addition of 100 nm insulin for periods up to 10 min. In some experiments, cells were stimulated with either 25 nm insulin, 20 nm EGF, 14 nm IGF-1, or 20% FBS for 15 min. These cells were washed in PBS and immersed in liquid nitrogen. The human HepG2 hepatoma cells and HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and McCoy's 5A medium, respectively, supplemented with 10% FBS, 2 mml-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. Transient Transfection Assays—HepG2 cells were cultured for 24 h until 60–80% confluence was reached. Transient transfection was performed according to the manufacturer's protocol for the use of LipofectAMINE 2000. In brief, empty expression vector (pcDNA3.1) or expression plasmid encoding HA-FLNaCT was mixed with the transfection reagent and directly added to the culture plates at a ratio of 6 μg/60-mm dish. Twenty four to 48 h later, cells were used for various experiments. Expression of HA-FLNaCT was analyzed in total cell lysates by immunoblotting with anti-HA antibody (Clontech, Palo Alto, CA). Cells were serum-starved for 18 h and incubated for 30 min with 200 μm orthovanadate prior to 100 nm insulin treatment for 10 min. Elk-1 Transactivation Assay—Transactivation of Elk-1 was examined by the PathDetect Elk-1 trans-Reporting System (Stratagene). In brief, HepG2 cells were cotransfected with pFR-Luc, pFA-Elk-1 and 0.2 μg of pCMV-β-galactosidase and either 3 μg of pcDNA3.1 or 3 μg of HA-FLNaCT as indicated. Serum-starved cells were left untreated or were stimulated with 100 nm insulin for 24 h. Elk-1 luciferase and β-galactosidase activities were measured using assay system kits from Promega according to the manufacturer's instructions, and the luciferase values were normalized to β-galactosidase. Detergent-free Isolation of Lipid Rafts—Isolation of lipid rafts was accomplished using a detergent-free sucrose gradient centrifugation method as described previously (19Yamamoto M. Toya Y. Schwencke C. Lisanti M.P. Myers Jr., M.G. Ishikawa Y. J. Biol. Chem. 1998; 273: 26962-26968Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). In brief, cells from 150-mm culture dishes were resuspended in 1 ml of 0.5 m sodium carbonate, pH 11, supplemented with a protease inhibitor mixture (Calbiochem), and homogenized by passing cells 15 times through a 23-gauge needle and two 10-s bursts of a sonicator probe on ice. The sucrose concentration in cell extracts was adjusted to 45% (w/w) by the addition of 1.8 volume of 70% (w/w) sucrose prepared in MBS (25 mm MES, pH 6.5, 0.15 m NaCl). At the bottom of an ultracentrifuge tube, 2 ml of the extracts were placed, followed by the addition of 5 ml of 35% (w/w) and 4 ml of 5% (w/w) sucrose prepared in MBS containing 0.25 m sodium carbonate. After centrifugation at 200,000 × g for 16 h at 4 °C in a Beckman SW41 rotor, a total of 11 fractions (1 ml each) were collected from the top of each gradient and used for immunoblotting. Immunoprecipitation and Immunoblotting—Unless otherwise indicated, cells were scraped in a lysis buffer (20 mm Hepes, pH 7.4, 137 mm NaCl, 100 mm NaF, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 0.02% NaN3, and 1 mm sodium orthovanadate) supplemented with protease inhibitor mixture (Calbiochem). After 30 min on ice, cell lysates were centrifuged (10,000 × g, 20 min, 4 °C), and the resulting clarified supernatants were collected. Equal amounts of solubilized proteins were incubated with anti-IR (1 μg of each clone), IRS-1 (5 μg), Shc (5 μg), or phosphotyrosine (5 μg) antibody at 4 °C overnight. Alternatively, to detect FLNa-IR association, cells were solubilized in TLB buffer (25 mm Tris·HCl, pH 8.0, 150 mm NaCl, 1% Triton X-100, 10% glycerol, 1 mm EDTA, 2 mm dithiothreitol, and protease inhibitor mixture), and the clarified supernatants were incubated with IR antibodies. Then, protein A/G-agarose (Oncogene Science) beads were added, and the incubation was continued at 4 °C for 4 h. The beads were pelleted by centrifugation and washed twice in lysis buffer and twice in 50 mm Hepes, pH 7.4, supplemented with 0.1% Triton X-100 before solubilization in Laemmli sample buffer (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar) supplemented with 5% 2-mercaptoethanol. In some experiments, cells were lysed directly in Laemmli sample buffer containing 5% 2-mercaptoethanol and 1 mm orthovanadate. After heating at 70 °C for 10 min, proteins were separated by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes. Detection of individual proteins was carried out by immunoblotting with specific primary antibodies and visualized by enhanced chemiluminescence. Signals were quantitated by densitometry using the ImageQuant software (Amersham Biosciences). Measurement of Thymidine Incorporation—M2 and A7 cells were grown to 75% confluence in 6-well cluster plates. The growth medium was replaced with serum-free MEM for 18 h. Cells were stimulated with insulin at the indicated concentrations (0, 1, 10, and 100 nm) for 18 h and then pulsed with [3H]thymidine (PerkinElmer Life Sciences), 2 μCi/ml, for 2 h at 37 °C. The cells were washed three times in ice-cold PBS, and DNA was precipitated with 10% trichloroacetic acid for 30 min on ice. After a rapid wash, trichloroacetic acid-precipitable material was dissolved in 0.5 ml of 1 m NaOH and then neutralized with HCl. Levels of [3H]thymidine incorporated into DNA were measured in a scintillation counter and expressed as counts/min. Values obtained without insulin stimulation were subtracted from the corresponding values obtained after treatment. GST Pull-down Experiments—HepG2 cell lysates (endogenous FLNa) or 5 μg of purified chicken gizzard FLNa (Research Diagnostics Inc.) were either incubated with anti-FLNa antibodies for 4 h followed by precipitation of the immune complexes with protein A/G-Sepharose beads or incubated for 16 h with 5 μg of GST or 5 μg of GST-IR β-subunit protein preimmobilized onto glutathione-agarose (Amersham Biosciences) at 4 °C in TLB buffer. After a series of washes in TLB buffer, the bound proteins were eluted in Laemmli sample buffer, separated by SDS-PAGE, and immunoblotted using anti-FLNa antibody. Fluorescence Microscopy—HepG2 cells grown on coverslips were fixed in fresh 4% paraformaldehyde in PBS for 10 min and permeabilized in 0.1% Triton X-100 in PBS for 10 min at room temperature. The cells were incubated with blocking buffer (8% bovine serum albumin in PBS) for 20 min at room temperature, washed in PBS supplemented with 0.5% bovine serum albumin and 0.05% Tween 20, and incubated with anti-IR β-subunit (1:100), FLNa (1:100), or HA (1:166) antibody for 16 h at 4 °C. After washing, cells were stained with Alexa Fluor secondary antibody (1:1000). For immunolocalization of F-actin, fixed cells were incubated with Alexa Fluor-conjugated phalloidin (1:20). Nuclear counterstaining was performed by incubating coverslips with Topro-3 in PBS for 5 min prior to mounting slides with Vectashield (Vector Laboratories). Images were acquired using a Zeiss LSM-410 inverted confocal microscope with a 63× oil-immersed objective and processed by using the Metamorph software (Universal Imaging Corp.). No fluorescent staining was observed when the primary antibody was omitted. Statistical Analysis—Quantitative data are presented as mean ± S.E., and differences between the means were determined by analysis of variance coupled to Fisher's least significance difference for multiple mean comparisons. A p value of <0.05 was considered significant. Statistical analyses were performed with the StatView statistical software program (SAS Institute, Inc., Cary, NC). Selective Impairment in Insulin Signaling in FLNa-expressing Cells —Insulin elicits rapid autophosphorylation of the IR and tyrosine phosphorylation of substrates, including IRS-1 and Shc proteins, in a number of cell lines. We examined FLNa-deficient M2 cells and FLNa-expressing subline A7 cells for their insulin responsiveness, and we found that both cell lines exhibited a comparable increase in insulin-induced IR autophosphorylation (Fig. 1A). Under basal conditions, cells that were pretreated with the protein-tyrosine phosphatase inhibitor orthovanadate had very low levels of IRS-1 and Shc tyrosine phosphorylation (Fig. 1, B and C). Addition of insulin elicited a significant increase in IRS-1 phosphorylation in vanadate-pretreated M2 and A7 cells (Fig. 1B). In contrast, the combination of insulin and vanadate strongly stimulated the levels of Shc tyrosine phosphorylation in M2 cells, while being rather ineffective in the FLNa-expressing A7 cells (Fig. 1C). PI3-kinase is an important enzyme implicated in insulin signal transduction through its interaction with tyrosine-phosphorylated IRS proteins (21White M.F. Recent Prog. Horm. Res. 1998; 53: 119-138PubMed Google Scholar). It is a heterodimeric enzyme encompassing a p85 regulatory subunit and a catalytic subunit (p110). We therefore examined the cosedimentation of p85 in IRS-1 immunoprecipitates to determine whether p85 association to IRS-1 might be affected by FLNa expression. As shown in Fig. 1B, insulin was able to stimulate recruitment of PI3-kinase to IRS-1 both in vanadate-pretreated M2 and A7 cells. Two of the major insulin signaling events initiated downstream of the IR is activation of the PI3-kinase/AKT pathway and Ras/MAPK cascade. To evaluate the involvement of FLNa expression on the regulation of these pathways, we examined the ability of insulin to activate AKT and p42/44 MAPK (also known as extracellular signal-regulated kinase, ERK 1/2) by immunoblotting cell lysates with phospho-specific antibodies (Fig. 2). The levels of phosphorylation of AKT at Ser-473, a modification required for its activation (22Hemmings B.A. Science. 1997; 275: 628-630Crossref PubMed Scopus (437) Google Scholar), were increased upon stimulation of M2 and A7 cells with insulin. In contrast to AKT, there was a marked attenuation in insulin-induced ERK phosphorylation in FLNa-expressing A7 cells (Fig. 2 upper panel, 9th versus 2nd lane). We then investigated whether the lack of FLNa plays a role in the phosphorylation of AKT and ERK in response to other stimuli. Both cell lines were equally responsive to EGF and serum; however, the increase in ERK phosphorylation levels induced by IGF-1 in M2 cells was blocked in FLNa-expressing A7 cells (Fig. 2, upper panel). Similar amounts of ERK protein were present in either cell line (Fig. 2, lower panel). Thus, expression of FLNa differentially affects ERK regulation in response to various stimuli. These results are consistent with a selective effect of FLNa in the signaling pathway used by insulin and IGF-1 to regulate p42/44 MAPK cascade. It is well recognized that phosphorylation of Shc by IR is necessary for activation of the Ras/MAPK pathway and mitogenesis (23Sasaoka T. Rose D.W. Jhun B.H. Saltiel A.R. Draznin B. Olefsky J.M. J. Biol. Chem. 1994; 269: 13689-13694Abstract Full Text PDF PubMed Google Scholar). In order to investigate the ability of FLNa to modulate mitogenic responses of insulin, we incubated M2 and A7 cells with increasing concentrations of the hormone for 18 h and measured DNA synthesis. FLNa-depleted M2 cells displayed an enhanced sensitivity to insulin when compared with FLNa-expressing A7 cells (Fig. 3). Both cell lines responded to serum (10%) stimulation by increasing thymidine incorporation to the same levels. The sensitivity of insulin-dependent activation of the Shc/MAPK cascade and the subsequent mitogenic responses to FLNa expression suggest specific perturbation of the signaling pathway proximal to the IR. Effect of FLNa Expression on Cellular Redistribution of Signaling Intermediates—Translocation of the Shc adaptor from the cytosol to lipid raft microdomains leads to MAPK activation (24Plyte S. Majolini M.B. Pacini S. Scarpini F. Bianchini C. Lanfrancone L. Pelicci P. Baldari C.T. Oncogene. 2000; 19: 1529-1537Crossref PubMed Scopus (46) Google Scholar). Moreover, the guanine nucleotide exchange factor SOS1 is prevalently cytosolic and must be brought to the cytosolic side of the plasma membrane in close juxtaposition to Ras to allow GDP/GTP exchange and Ras activation (25Chardin P. Camonis J.H. Gale N.W. van Aelst L. Schlessinger J. Wigler M.H. Bar-Sagi D. Science. 1993; 260: 1338-1343Crossref PubMed Scopus (658) Google Scholar). To establish whether expression of FLNa could influence the ability of signaling intermediates to be redistributed upon insulin stimulation, lipid rafts were isolated by sucrose gradient centrifugation from lysates of M2 and A7 cells and immunoblotted with specific antibodies. Preliminary studies showed that both cell types contained similar levels of signaling molecules, such as Shc, SOS1, ERK, and c-Src (Fig. 4A). Light fractions, enriched in rafts, and heavy fractions, containing most soluble proteins, were first tested by immunoblot with anti-SOS1 antibodies. As shown in Fig. 4B (panels I and III), endogenous SOS1 was found only in the heavy fractions of unstimulated cells, demonstrating that SOS1 is predominantly in the cytosol. However, addition of insulin resulted in inducible localization of SOS1 in the light fractions of FLNa-depleted M2 cells but not from FLNa-expressing A7 cells (Fig. 4B, panel II versus IV). The latter cells were found to have also impaired translocation of Shc, ERK, and phosphoactive ERK to the membrane rafts following insulin treatment (Fig. 4C). Of importance, FLNa expression did not abrogate the levels of c-Src, and presumably of other raft-associated proteins, in these specialized membrane microdomains (Fig. 4C, bottom panel). These results are consistent with the hypothesis that FLNa plays an important role in insulin-dependent Shc/MAPK cascade signaling at a step proximal to the IR. The IR Is an FLNa-interacting Protein—FLNa interacts with a number of cell surface proteins and intracellular signaling molecules (6Stossel T.P. Condeelis J. Cooley L. Hartwig J.H. Noegel A. Schleicher M. Shapiro S.S. Nat. Rev. Mol. Cell Biol. 2001; 2: 138-145Crossref PubMed Scopus (827) Google Scholar). We examined whether IR could form a complex with FLNa in intact cells. Our data demonstrated that IR was associated constitutively with FLNa in A7 cells but not in M2 cells that lack FLNa (Fig. 5A, right panels). Insulin treatment did not cause a significant change in the formation of the IR-FLNa complex in A7 cells. Similar results were obtained in HEK293 cells and HepG2 hepatoma whereby IR-FLNa association was detected under basal and insulin-stimulated conditions (Fig. 5A, left panels). In light of previous evidence supporting the actin-binding properties of FLNa, we sought to examine the role of cytoskeletal organization in the regulation of IR-FLNa association. To this end, HepG2 hepatoma were treated with cytochalasin D, an agent that causes depolymerization of actin (26Tsakiridis
Referência(s)