Type I Transforming Growth Factor β Receptor Binds to and Activates Phosphatidylinositol 3-Kinase

We have examined the interaction of transforming growth factor (TGF)β receptors with phosphatidylinositol 3-(PI3) kinase in epithelial cells. In COS7 cells, treatment with TGFβ increased PI3 kinase activity as measured by the ability of p85-associated immune complexes to phosphorylate inositides in vitro. Both type I and type II TGFβ receptors (TβR) associated with p85, but the association of TβRII appeared to be constitutive. The interaction of TβRI with p85 was induced by treatment with TGFβ. The receptor association with PI3 kinase was not direct as 35S-labeled rabbit reticulocyte p85 did not couple with fusion proteins containing type I and type II receptors. A kinase-dead, dominant-negative mutant of TβRII blocked ligand-induced p85-TβRI association and PI3 kinase activity. In TβRI-null R1B cells, TGFβ did not stimulate PI3 kinase activity. This stimulation was restored upon reconstitution of TβRI by transfection. In R1B and NMuMG epithelial cells, overexpression of a dominant active mutant form of TβRI markedly enhanced ligand-independent PI3 kinase activity, which was blocked by the addition of the TβRI kinase inhibitor LY580276, suggesting a causal link between TβRI function and PI3 kinase. Overexpressed Smad7 also prevented ligand-induced PI3 kinase activity. Taken together, these data suggest that 1) TGFβ receptors can indirectly associate with p85, 2) both receptors are required for ligand-induced PI3 kinase activation, and 3) the activated TβRI serine-threonine kinase can potently induce PI3 kinase activity. We have examined the interaction of transforming growth factor (TGF)β receptors with phosphatidylinositol 3-(PI3) kinase in epithelial cells. In COS7 cells, treatment with TGFβ increased PI3 kinase activity as measured by the ability of p85-associated immune complexes to phosphorylate inositides in vitro. Both type I and type II TGFβ receptors (TβR) associated with p85, but the association of TβRII appeared to be constitutive. The interaction of TβRI with p85 was induced by treatment with TGFβ. The receptor association with PI3 kinase was not direct as 35S-labeled rabbit reticulocyte p85 did not couple with fusion proteins containing type I and type II receptors. A kinase-dead, dominant-negative mutant of TβRII blocked ligand-induced p85-TβRI association and PI3 kinase activity. In TβRI-null R1B cells, TGFβ did not stimulate PI3 kinase activity. This stimulation was restored upon reconstitution of TβRI by transfection. In R1B and NMuMG epithelial cells, overexpression of a dominant active mutant form of TβRI markedly enhanced ligand-independent PI3 kinase activity, which was blocked by the addition of the TβRI kinase inhibitor LY580276, suggesting a causal link between TβRI function and PI3 kinase. Overexpressed Smad7 also prevented ligand-induced PI3 kinase activity. Taken together, these data suggest that 1) TGFβ receptors can indirectly associate with p85, 2) both receptors are required for ligand-induced PI3 kinase activation, and 3) the activated TβRI serine-threonine kinase can potently induce PI3 kinase activity. Transforming growth factor β (TGFβ) 1The abbreviations used are: TGFβ, transforming growth factor β; TβRI, type I TGFβ receptor; TβRII, type II TGFβ receptor; PI3 kinase, phosphatidylinositol 3-kinase; Erk, extracellular signal-regulated kinase; Jnk, c-Jun N-terminal kinase; MAPKs, mitogen-activated protein kinases; PI3P, phosphatidylinositol-3 monophosphate; GST, glutathione S-transferase; GSH, glutathione; PI, phosphoinositides; HA, hemagglutinin; EGFP, enhanced green fluorescent protein; SH2, Src homology. binds to a heteromeric complex of transmembrane serine-threonine kinases, the type I and II TGFβ receptors (TβRI or Alk5 and TβRII). Following ligand binding to TβRII, the type I receptor is recruited to the ligand-receptor complex where the constitutively active TβRII transactivates TβRI (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). Activated TβRI phosphorylates the receptor-specific Smad2 and Smad3, which then associate with Smad4 and, as a heteromeric complex, translocate to the nucleus where they regulate the transcription of TGFβ target genes (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). The Smad signaling pathway mediates the antiproliferative effect of TGFβ in epithelial cells (2Liu X. Sun Y. Constantinescu S.N. Karam E. Weinberg R.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10669-10674Crossref PubMed Scopus (331) Google Scholar, 3Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (916) Google Scholar) and is the best characterized. Several non-Smad pathways have also been implicated in mediating the cellular effects of TGFβ. These include the extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 (PI3) kinase, and the family of Rho GTPases (4Wakefield L.M. Roberts A.B. Curr. Opin. Genet. Dev. 2002; 12: 22-29Crossref PubMed Scopus (732) Google Scholar, 5Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4324) Google Scholar). Activated PI3 kinase increases the formation of intracellular 3′-phosphorylated inositol lipids, signal transducers that are essential for the regulation of cell cycle progression, glucose metabolism, cell motility, epithelial-to-mesenchymal transition, and apoptosis among others (6Vanhaesebroeck B. Waterfield M.D. Exp. Cell Res. 1999; 253: 239-254Crossref PubMed Scopus (764) Google Scholar, 7Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5171) Google Scholar). In Swiss 3T3 cells, TGFβ stimulates PI3 kinase activity, as measured by the ability of immune complexes precipitated with an antibody against p85, the regulatory subunit of PI3 kinase, to induce the formation of phosphatidylinositol-3 monophosphate (PI3P) in vitro (8Higaki M. Shimokado K. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2127-2132Crossref PubMed Scopus (61) Google Scholar). In mammary epithelial cells, TGFβ induces epithelial-tomesenchymal transition and cell motility as well as phosphorylation and activation of the PI3 kinase-dependent Akt serinethreonine kinase. These responses are blocked by LY294002, a small molecule inhibitor of the p110 catalytic subunit of PI3 kinase (9Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. J. Biol. Chem. 2000; 275: 36803-36810Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar). In mesenchymal and epithelial cells, TGFβ-mediated protection from apoptosis is blocked by LY294002 and/or expression of dominant-negative, kinase-dead Akt (10Shin I. Bakin A.V. Rodeck U. Brunet A. Arteaga C.L. Mol. Biol. Cell. 2001; 12: 3328-3339Crossref PubMed Scopus (153) Google Scholar, 11Horowitz J.C. Lee D.Y. Waghray M. Keshamouni V.G. Thomas P.E. Zhang H. Cui Z. Thannickal V.J. J. Biol. Chem. 2004; 279: 1359-1367Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 12Muraoka R.S. Kurokawa H. Koh Y. Forbes J.T. Roebuck L.R. Barcellos-Hoff M.H. Moody S.E. Chodosh L.A. Arteaga C.L. Cancer Res. 2004; 64: 9002-9011Crossref PubMed Scopus (141) Google Scholar). Inhibition of PI3 kinase reverses the fibroblastoid phenotype of TGFβ-treated Ras-transformed hepatocytes to an epithelial phenotype (13Gotzmann J. Huber H. Thallinger C. Wolschek M. Jansen B. Schulte-Hermann R. Beug H. Mikulits W. J. Cell Sci. 2002; 115: 1189-1202PubMed Google Scholar). Furthermore, PI3 kinase inhibition abrogates both basal and TGFβ-induced motility in mammary cancer cells (9Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. J. Biol. Chem. 2000; 275: 36803-36810Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar). Taken together, these data suggest that PI3 kinase is a major effector pathway of the transforming effects of TGFβ in epithelial cells. Therefore, in this study, we have examined the initial mechanisms of TGFβ-induced activation of PI3 kinase in epithelial cells. Cell Lines and Antibodies—COS7 and NMuMG cells were purchased from American Type Culture Collection (ATCC). R1B cells were provided by Harold Moses (Vanderbilt University, Nashville, TN). COS7 and R1B cells were maintained in Dulbecco's Modified Essential Medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone). NMuMG cells were maintained in 10% fetal bovine serum-Dulbecco's modified Eagle's medium with 10 μg/ml insulin. We utilized the following antibodies: p85 (Upstate Biotechnology), Smad2/3 (Transduction Laboratories), hemagglutinin (HA) rabbit polyclonal and TβRI (Santa Cruz Biotechnology), FLAG epitope (Sigma), Ser-473 P-Akt, total Akt, P-MAPK, total MAPK (Erk42/44), and P-Smad2 (Cell Signaling). Human recombinant TGFβ1 was obtained from R 8: 1243-1252Abstract Full Text Full Text PDF PubMed Google Scholar). The wild-type TβRI-HA construct, from Kohei Miyazono (Japanese Foundation for Cancer Research, Tokyo), was subcloned into the retroviral expression vector pBMN-IRES-EGFP (provided by Garry Nolan, Stanford University, Palo Alto, CA) (15Grignani F. Kinsella T. Mencarelli A. Valtieri M. Riganelli D. Lanfrancone L. Peschle C. Nolan G.P. Pelicci P.G. Cancer Res. 1998; 58: 14-19PubMed Google Scholar). Cells were transfected with plasmids using Fu-GENE 6 reagent (Roche Applied Sciences) according to the manufacturer's instructions. Retroviruses were prepared by transfection of 293T cells with 15 μg of DNA/100-mm dish of three plasmids encoding gag/pol, VSV-G, and the target construct (in 4:3:8 ratios, respectively). Supernatants from cells were collected for 2 days and combined, filtered through 0.4-μm filters, and stored in aliquots at -80 °C. R1B cells were infected with supernatant containing retroviruses in the presence of 8 μg/ml Polybrene (Sigma) as described previously (16Yee J.K. Miyanohara A. LaPorte P. Bouic K. Burns J.C. Friedmann T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9564-9568Crossref PubMed Scopus (445) Google Scholar). Three days after transduction of R1B cells with pBMN-IRES-EGFP, EGFP-positive cells were selected by flow cytometry. Under these conditions, >95% of selected cells expressed GFP at the time of any of our experiments. The HA-Alk5T204D and Flag-Smad7 adenoviral constructs were also from Dr. Kohei Miyazono. Stocks of recombinant adenoviruses were generated in 293T cells and titered utilizing the Takara assay (Takara Biomedicals, Tokyo, Japan). Cells were then infected with these or with a control β-galactosidase adenovirus at an equivalent multiplicity of infection as described previously (17Dumont N. Bakin A.V. Arteaga C.L. J. Biol. Chem. 2003; 278: 3275-3285Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Immunoprecipitation and Immunoblot Analysis—Cells were washed twice with phosphate-buffered saline and lysed in 1% Nonidet P-40 buffer (1% Nonidet P-40, 120 mm NaCl, and 50 mm Tris-HCl, pH 7.4, protease inhibitor mixture (Roche Applied Science), 20 mm NaF, 1 mm Na3VO4) on ice for 20 min. Cell lysates were clarified by centrifugation at 14,000 rpm at 4 °C for 20 min, and protein concentrations were then determined by the BCA method (Pierce). Equal amounts of cell lysates were incubated overnight at 4 °C with primary antibodies. Immune complexes were collected with protein G-Sepharose 4B or protein A-Sepharose 4B (both from Sigma) at 4 °C for 3 h. The precipitates were pelleted by centrifugation and washed three with with buffer mm Tris-HCl, pH 7.4, and with buffer mm Tris-HCl, pH The were in buffer containing 10% and by μg of total cell lysates were by and to were blocked with in saline containing at for 1 h. The were incubated with primary antibody in 4 °C for washed three with for 1 incubated for 2 with or antibody with and then washed three with for an 1 h. were using an enhanced as described (17Dumont N. Bakin A.V. Arteaga C.L. J. Biol. Chem. 2003; 278: 3275-3285Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). expression for glutathione fusion and from and (provided by Rik were into The were induced by mm for at °C. E. were washed with buffer mm pH mm NaCl, 1 mm and incubated in buffer containing μg of for 15 on mm and were to the E. which were lysates were in 10 μg of and 8 mm and incubated for 2 at 4 °C by centrifugation at for min. fusion proteins were by binding to glutathione 4B at for 1 three with phosphate-buffered saline containing protease The of proteins on was by and against bovine serum (Sigma) after the with were between fusion proteins and in vitro p85 p85 was generated with a reticulocyte also containing and 35S-labeled 10 of 35S-labeled p85 protein and amounts of or fusion proteins were incubated for 2 at 4 °C with The precipitates were pelleted by centrifugation and washed three with 1% Nonidet P-40 with buffer and with The were in by and by PI3 kinase catalytic activity was measured in vitro as described previously C.L. 1997; PubMed Scopus Google Scholar). In cells were lysed with 1% Nonidet P-40 in buffer A mm NaCl, 20 mm Tris-HCl, pH 7.4, 1 mm 1 mm and mm Na3VO4) and a protease inhibitor were precipitated with a p85 antibody and protein A-Sepharose 2 in 1% Nonidet P-40 in buffer A and 2 in assay buffer mm mm NaCl, mm the precipitates were in of assay buffer containing 10 of 2 Sigma) and 10 of with 10 of activity in mm pH and 50 mm Following for 10 at the was by the addition of of 1 and of The were and by using with 1% in a of The of into was by p85, the of PI3 Both TβRII and COS7 cells, addition of TGFβ PI3 kinase activity in a as measured by the ability of immune complexes precipitated with p85 to stimulate formation of 3′-phosphorylated inositol in vitro. This was at 6 and with phosphorylation of the serine-threonine kinase Akt in Ser-473 The of ligand-induced Smad2 phosphorylation were with after treatment with TGFβ for 1 h. p85 with TGFβ we COS7 cells with encoding p85 and TβRI or TβRII by with receptor antibodies. p85 was in TβRI and TβRII from cells with the of this binding is by TGFβ, COS7 cells were with TGFβ for The binding between p85 and TβRI was 1 after TGFβ and this association was with of Smad2 and Akt phosphorylation binding between p85 and TβRII was not by TGFβ in these cells a ligand-induced phosphorylation of Smad2 and Akt was this receptor association was we rabbit reticulocyte 35S-labeled p85 with and with fusion proteins Under these conditions, we were to p85 from the receptor that the p85-TβRI and as by the in transfected cells A and were not TβRII and TβRI for of PI3 TβRII function is required for ligand-induced activation of PI3 kinase, we COS7 cells with p85 and TβRI with or a vector encoding a TβRII mutant in which the at has been to (14Oft M. Heider K.H. Beug H. Curr. Biol. 1998; 8: 1243-1252Abstract Full Text Full Text PDF PubMed Google Scholar). The expression of the construct prevented ligand-induced association of p85 with TβRI In cells with p85 and TβRI, we were to an in TGFβ-induced PI3 kinase activity associated with p85 precipitates not We that these conditions, the of the p85 protein is not associated with TβRI, and the p85-associated catalytic activity not TGFβ PI3 kinase activity. Therefore, we transfected TβRI p85 but with or The expression of the kinase-dead type II receptor a in PI3 kinase activity in vitro This is from the of ligand-induced activation in COS7 cells In COS7 cells transfected with TβRI, but not with TβRII, TGFβ increased PI3 kinase activity at 1 h. We did not any PI3 kinase activity in TβRI TβRI was required for ligand-induced activation of PI3 kinase, we used R1B cells, a of cells, which type I receptors J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). R1B cells were with pBMN-IRES-EGFP or retroviral EGFP-positive cells were by flow cytometry. In our >95% of cells used expressed In cells but not in cells with the control treatment with TGFβ induced PI3 kinase activity as well as Smad2 and Akt phosphorylation TβRI PI3 p85 can TβRII we determined overexpression of TβRII was for ligand-induced PI3 kinase and and these with the of activation in COS7 cells In COS7 cells transfected TβRII, TGFβ induced PI3 kinase activity and Ser-473 at 6 suggesting that an of type II receptor did not PI3 kinase activation with cells. We were to any PI3 kinase activity in immune complexes precipitated with TβRII The that in cells transfected p85 and TβRI, the association of p85 and type I receptors was after 1 of treatment with TGFβ in COS7 and R1B cells transfected TβRI, ligand-induced activation of PI3 kinase was after 1 of treatment and suggesting that TβRI can the activation of PI3 kinase. Therefore, to TβRI can PI3 kinase, we NMuMG and R1B cells with an adenovirus encoding a mutant of TβRI A of with to activation of the type I receptor serine-threonine kinase to signal in the of ligand R. J. EMBO J. PubMed Scopus Google Scholar). In both cells, transduction with a mutant but not with a β-galactosidase control adenovirus in a in p85-associated PI3 kinase activity as well as Smad2 phosphorylation active Alk5 was associated with the enhanced PI3 kinase activity we utilized small molecule TβRI serine-threonine kinase inhibitor LY580276 T. N. S. L. Zhang F. Yingling Chem. 2004; PubMed Scopus Google Scholar). LY580276 is a that is for TβRI to TβRII and a of with an activity in cells and The of LY580276 in has been which binding at the J. Lee Zhang F. Yingling Curr. Opin. Dev. 2004; Google Scholar). with LY580276 for blocked PI3 kinase activation and Smad2 phosphorylation a causal association between active Alk5 signaling and the of PI3 kinase catalytic activity. Smad7 of PI3 the activation of PI3 kinase by TβRI Smad we blocked Smad signaling with the Smad7 H. S. Y. J. D. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. A. F. Heldin C.H. Heldin ten Dijke P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Smad7 with activated TβRI the association with phosphorylation and activation of Overexpressed adenoviral Smad7 blocked PI3 kinase activity as well as Smad2 phosphorylation We have examined TGFβ receptors with and/or PI3 kinase in epithelial cells. TGFβ increased PI3 kinase activity as measured by the ability of p85-associated immune complexes to phosphorylate inositides in vitro. The of ligand-induced PI3 kinase activation were the phosphorylation of Smad2 with 6 after the addition of TGFβ. Both type I and type II receptors associated with p85, but the association of TβRII appeared as was not enhanced by the the interaction of TβRI with p85 was markedly enhanced by treatment with TGFβ for 1 h. This receptor association with PI3 kinase was not rabbit reticulocyte p85 did not associate with fusion proteins containing type I and type II receptors. In we were to any PI3 kinase activity in TβRI and TβRII precipitates from cells. This is with a using in which PI3 kinase activity was not with a TβRII antibody (8Higaki M. Shimokado K. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2127-2132Crossref PubMed Scopus (61) Google Scholar). These and the of of PI3 kinase catalytic activity by TGFβ suggest the presence of signaling between TGFβ receptors and PI3 kinase. TβRII overexpression did not TGFβ-induced or PI3 kinase activity, a kinase-dead, dominant-negative mutant of TβRII blocked ligand-induced p85-TβRI association and PI3 kinase activity suggesting that a type II receptor is required for TGFβ-mediated activation of PI3 kinase. These data also that in with in the TGFβ not be to PI3 kinase and to by this effector In TβRI-null R1B cells, TGFβ did not stimulate PI3 kinase activity. this stimulation was restored upon reconstitution of TβRI by transfection. the overexpression of TβRI ligand-induced catalytic activity with 1 after the addition of TGFβ. that the serine-threonine kinase activity of TβRI can PI3 kinase, we R1B and NMuMG epithelial cells with a dominant active mutant form of In both cell the expression of markedly enhanced ligand-independent PI3 kinase activity, which was blocked by the addition of the TβRI kinase inhibitor LY580276, suggesting a causal link between TβRI function and PI3 kinase. which binds TβRI, prevented ligand-induced PI3 kinase activity as well as Smad2 that TβRI can associate with and PI3 kinase. this causal between TβRI function and TGFβ-mediated activation of PI3 kinase, is not TβRI stimulates this activity. The of with ligand is with that in growth Swiss 3T3 (8Higaki M. Shimokado K. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2127-2132Crossref PubMed Scopus (61) Google Scholar) and cells J. R. Waterfield M.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) where the p85-associated PI3 kinase activity is increased of cellular PI3 kinase is activated by or receptors A.B. Google Scholar, P. E. Nature. PubMed Scopus Google Scholar, E. R. R. E. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This of activation the of PI3 kinase that is by a growth In cells, for TGFβ can induce a in PI3 kinase in the presence of growth factor R. A. R.A. J. 1997; PubMed Google Scholar). with this overexpression of p85 in COS7 cells the in p85-associated PI3 kinase activation in to an ligand not the of p85-associated PI3 kinase in cells with the constitutively active which was blocked by a inhibitor of the type I receptor kinase, causal TβRI and PI3 kinase (17Dumont N. Bakin A.V. Arteaga C.L. J. Biol. 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We were to any PI3 kinase activity in from TGFβ-treated cells not suggesting that PI3 kinase activation is not by Therefore, we this is the of PI3 kinase activation induced by a serine-threonine kinase in epithelial cells. to be determined TGFβ-induced PI3 kinase activity is among epithelial cells. in as our data PI3 kinase activation signaling the expression of TGFβ can or this signaling pathway as well as the and time of this we have not ligand-induced activation of Akt in and mammary epithelial cells. Akt activity is PI3 this suggest that activation of PI3 kinase by TGFβ is cell The to these