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Requirement of BMP-2-induced Phosphatidylinositol 3-Kinase and Akt Serine/Threonine Kinase in Osteoblast Differentiation and Smad-dependent BMP-2 Gene Transcription*

  • Nandini Ghosh-Choudhury
    Correspondence
    To whom correspondence should be addressed
    Footnotes
    Affiliations
    Department of Pathology, University of Texas Health Sciences Center, San Antonio, Texas 78229
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  • Sherry L. Abboud
    Footnotes
    Affiliations
    Department of Pathology, University of Texas Health Sciences Center, San Antonio, Texas 78229
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  • Riko Nishimura
    Affiliations
    Osaka University Faculty of Dentistry, Osaka 565-0871, Japan
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  • Anthony Celeste
    Affiliations
    Genetics Institute, Cambridge, Massachusetts 01810
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  • Lenin Mahimainathan
    Affiliations
    Department of Medicine, University of Texas Health Sciences Center, San Antonio, Texas 78229
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  • Goutam Ghosh Choudhury
    Affiliations
    Department of Medicine, University of Texas Health Sciences Center, San Antonio, Texas 78229

    Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System, San Antonio, Texas 78229
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  • Author Footnotes
    * This study was supported in part by the Dept. of Veterans Affairs Medical Research Service Merit Review Award, the Research Excellence Area Program (REAP) Award, National Institutes of Health Grant RO1 DK55815 (NIDDK) (to G. G. C.), and Dept. of Defense Breast Cancer Award DAMD17-99-1-9400 (to N. G. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Supported by a grant from the San Antonio Area foundation and an institutional Howard Hughes grant.
    ‖ Supported by National Institutes of Health Grant AR-42306.
Open AccessPublished:September 06, 2002DOI:https://doi.org/10.1074/jbc.M205053200
      The mechanism by which bone morphogenetic protein-2 (BMP-2) induces osteoblast differentiation is not precisely known. We investigated the involvement of the phosphatidylinositol (PI) 3-kinase/Akt signal transduction pathway in modulation of this process. BMP-2 stimulated PI 3-kinase activity in osteogenic cells. Inhibition of PI 3-kinase activity with the specific inhibitor Ly-294002 prevented BMP-2-induced alkaline phosphatase, an early marker of osteoblast differentiation. Expression of dominant-negative PI 3-kinase also abolished osteoblastic induction of alkaline phosphatase in response to BMP-2, confirming the involvement of this lipid kinase in this process. BMP-2 stimulated Akt serine/threonine kinase activity in a PI 3-kinase-dependent manner in osteoblast precursor cells. Inhibition of Akt activity by a dominant-negative mutant of Akt blocked BMP-2-induced osteoblastic alkaline phosphatase activity. BMP-2 stimulates its own expression during osteoblast differentiation. Expression of dominant-negative PI 3-kinase or dominant-negative Akt inhibited BMP-2-induced BMP-2 transcription. Because all the known biological activities of BMP-2 are mediated by transcription via BMP-specific Smad proteins, we investigated the involvement of PI 3-kinase in Smad-dependent BMP-2 transcription. Smad5 stimulated BMP-2 transcription independent of addition of the ligand. Dominant-negative PI 3-kinase or dominant-negative Akt inhibited Smad5-dependent transcription of BMP-2. Furthermore dominant-negative Akt inhibited translocation of BMP-specific Smads into nucleus. Together these data provide the first evidence that activation of BMP receptor serine/threonine kinase stimulates the PI 3 kinase/Akt pathway and define a role for this signal transduction pathway in BMP-specific Smad function during osteoblast differentiation.
      BMP
      bone morphogenetic protein
      BMPR
      BMP receptor
      PI
      phosphatidylinositol
      DN
      dominant-negative
      HA
      hemagglutinin
      TGF
      transforming growth factor
      GFP
      green fluorescence protein
      ERK
      extracellular signal-regulated kinase
      pNPP
      p-nitrophenylphosphate
      LUC
      luciferase
      Bone morphogenetic proteins (BMPs),1 a group of polypeptides within the transforming growth factor (TGF)-β superfamily, were originally identified by their ability to induce endochondral bone formation in ectopic extraskeletal sites in vivo (
      • Reddi A.H.
      ). BMPs stimulate differentiation of pluripotent mesenchymal cells into the osteogenic lineage and enhance the differentiated function of osteoblasts. Among many other BMPs, BMP-2 induces differentiation of preosteoblasts into mature osteoblasts by regulating signals that stimulate a specific transcriptional program required for bone formation (
      • Sakou T.
      ,
      • Ghosh-Choudhury N.
      • Harris M.A.
      • Feng J.Q.
      • Mundy G.R.
      • Harris S.E.
      ).
      Similar to TGF-β, BMPs exert their effect via type I and type II transmembrane serine/threonine kinase receptors (,
      • Massague J.
      ). The type II receptor binds the ligand with high affinity. The type I receptors, Alk3 (BMPRIA) and Alk6 (BMPRIB) are mainly responsible to transduce the signal, although they have weak BMP binding properties (
      • Kawabata M.
      • Imamura T.
      • Miyaxono K.
      ). Binding of BMP to its receptors induces phosphorylation of the type I receptor in its GS domain and recruitment of receptor-specific Smads (,
      • Massague J.
      ,
      • Heldin C.-H.
      • Myazono K.
      • ten Dijke P.
      ). Upon phosphorylation, Smad1, or one of its close homologs Smad5 and Smad8, heterodimerizes with the common Smad, Smad4. This complex translocates to the nucleus where it associates with transcriptional coactivators and acts as a transcription factor to regulate tissue or cell type-specific genes required for the divergent biological functions of BMPs (,
      • Massague J.
      ,
      • Heldin C.-H.
      • Myazono K.
      • ten Dijke P.
      ). In osteogenic cells, BMP-specific Smads stimulate expression of Cbfa1, the only known osteoblast-specific transcription factor to induce genes required for mature osteoblast differentiation and maintenance (
      • Karsenty G.
      ,
      • Ducy P.
      • Schinke T.
      • Karcenty G.
      ).
      Differentiation in response to extracellular factors depends on the action of kinases including both tyrosine and serine/threonine kinases (
      • Bonni A.
      • Sun Y.
      • Nadal-Vicens M.
      • Bhatt A.
      • Frank D.A.
      • Rozovsky I.
      • Stahl N.
      • Yancopoulos G.D.
      • Greenberg M.E.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ). In the case of neuronal differentiation, the tyrosine kinase activity of nerve growth factor (NGF) receptor and its downstream target ERK1/2-type of mitogen-activated serine/threonine protein kinase are essential (
      • Fukuda M.
      • Gotoh Y.
      • Tachibana T.
      • Dell K.
      • Hattori S.
      • Yoneda Y.
      • Nishida E.
      ). Recently a critical role for a lipid kinase, phosphatidylinositol 3-kinase (PI 3-kinase), has been suggested in muscle and adipocyte differentiation (
      • Jiang B-H.
      • Zheng J.Z.
      • Vogt P.K.
      ,
      • Sakaue H.
      • Ogawa W.
      • Matsumoto M.
      • Kuroda S.
      • Takata M.
      • Suginoto T.
      • Spiegelman B.M.
      • Kasuga M.
      ). Activation of PI 3-kinase is often associated with increased tyrosine phosphorylation induced by growth and differentiation factors (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). PI 3-kinase functions as the focal point in cellular signaling leading to cell growth, regulating cytoskeletal structure, and preventing apoptosis (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ,
      • Toker A.
      • Cantley L.C.
      ,
      • Franke T.F.
      • Cantley L.C.
      ). Involvement of PI 3-kinase has recently been shown to regulate some of the biological properties of TGF-β such as epithelial-mesenchymal transition and matrix protein expansion (
      • Bakin A.V.
      • Tomlinson A.K.
      • Bhowmick N.A.
      • Moses H.L.
      • Arteaga C.L.
      ). However, the role of PI 3-kinase has not been investigated in osteogenesis, especially in response to BMPs. In this study, we show that BMP-2 stimulates tyrosine phosphorylation and PI 3-kinase activity in osteogenic cells. We demonstrate the requirement of PI-3 kinase and its downstream target, Akt serine/threonine kinase, for BMP-2-induced expression of an osteoblast differentiation marker alkaline phosphatase, and for BMP-2 transcription. Finally we demonstrate that a cross-talk exists between BMP-specific Smad and PI 3-kinase/Akt pathway to induce transcription of BMP-2, a necessary growth and differentiation factor for osteogenic cells.

      DISCUSSION

      These studies represent the first demonstration of activation of the PI 3-kinase/Akt pathway in response to the osteogenic factor BMP-2. We demonstrate that BMP-2-induced expression of alkaline phosphatase, an enzyme expressed during osteoblast differentiation of progenitor cells into mature osteoblast, requires activation of PI 3-kinase and its downstream target Akt serine/threonine kinase. Our data also provide the first evidence that PI 3-kinase and Akt modulate autoregulation of BMP-2 gene transcription and that a cross-talk exists between the BMP-specific Smad and PI 3-kinase/Akt signaling pathway for regulation of BMP-2 transcription.
      Activation of PI 3-kinase regulates cellular processes including proliferation, migration, secretion, endocytosis, and protein transport (
      • Toker A.
      • Cantley L.C.
      ). Recently a role of PI 3-kinase has been shown in TGF-β-induced epithelial and endothelial cell survival and epithelial to mesenchymal transition (
      • Bakin A.V.
      • Tomlinson A.K.
      • Bhowmick N.A.
      • Moses H.L.
      • Arteaga C.L.
      ,
      • Shin I.
      • Bakin A.V.
      • Rodeck U.
      • Brunet A.
      • Arteaga C.L.
      ,
      • Vinals F.
      • Pouyssegur J.
      ) indicating that activation of serine/threonine kinase receptor utilizes this central lipid kinase as one of the signaling mechanisms similar to receptor tyrosine kinases. Recently PI 3-kinase has been implicated in myogenic differentiation (
      • Jiang B-H.
      • Zheng J.Z.
      • Vogt P.K.
      ,
      • Kaliman P.
      • Vinals F.
      • Testar X.
      • Palacin M.
      • Zorzano A.
      ,
      • Pinset C.
      • Garcia A.
      • Rousse S.
      • Dubois C.
      • Montarras D.
      ). Also insulin-induced adipocyte differentiation utilizes activation of PI 3-kinase pathway (
      • Kulik G.
      • Klippel A.
      • Weber M.J.
      ,
      • Yao R.
      • Cooper G.M.
      ). We have shown here that the osteogenic differentiation factor BMP-2 stimulates PI 3-kinase activity (Fig. 1). Increased PI 3-kinase activity is often associated with receptor and non-receptor tyrosine kinase-mediated signal transduction in which activation of this lipid kinase is a result of its association with specific tyrosine-phosphorylated proteins (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). In this report we demonstrate that activation of the BMP receptor results in increased tyrosine phosphorylation of proteins leading to association of the regulatory subunit of PI 3-kinase with tyrosine-phosphorylated proteins (Fig. 1, B and C). Also, we document BMP-2 induced PI 3-kinase activity in the tyrosine-phosphorylated protein fraction (Fig. 1D). Additionally we demonstrate that both BMPRIA and BMPRIB are involved in activation of PI 3-kinase in the tyrosine-phosphorylated protein fraction (Fig. 5). However, we were unable to detect any association of PI 3-kinase with the BMPRI in the BMPRI immunoprecipitates (data not shown). These data indicate that not only receptor tyrosine kinases, but serine/threonine kinase receptor utilizes the similar tyrosine phosphorylation mechanism to stimulate PI 3-kinase activity. It is evident from our data that the PI 3-kinase activity in p85 immunoprecipitates is somewhat less (Fig.1A) than that in anti-phosphotyrosine immunoprecipitates (Fig. 1D). One reason for this may be that p85 antibody recognizes the total pool of PI 3-kinase present in the cell whereas anti-phosphotyrosine antibody recognizes only the activated form of PI 3-kinase, which is associated with tyrosin-phosphorylated proteins in response to BMP-2.
      Differentiation of cells is always associated with induction of specific protein markers resulting from cell- and differentiation-specific transcriptional programs. Thus a hallmark of osteoblast differentiation is expression of alkaline phosphatase, which is induced early in the differentiation process (
      • Ghosh-Choudhury N.
      • Harris M.A.
      • Feng J.Q.
      • Mundy G.R.
      • Harris S.E.
      ,
      • Ghosh-Choudhury N.
      • Windle J.J.
      • Koop B.A.
      • Harris M.A.
      • Guerrero D.L.
      • Mundy G.R.
      • Harris S.E.
      ,
      • Ghosh-Choudhury N.
      • Harris M.A.
      • Wozney J.
      • Mundy G.R.
      • Harris S.E.
      ). We showed previously that BMP-2 stimulated alkaline phosphatase mRNA and protein expression in 2T3 cells during the initiation of differentiation (
      • Ghosh-Choudhury N.
      • Harris M.A.
      • Feng J.Q.
      • Mundy G.R.
      • Harris S.E.
      ,
      • Ghosh-Choudhury N.
      • Windle J.J.
      • Koop B.A.
      • Harris M.A.
      • Guerrero D.L.
      • Mundy G.R.
      • Harris S.E.
      ). Using a PI 3-kinase inhibitor, we have now demonstrated that activation of this lipid kinase is essential for BMP-2-induced alkaline phosphatase activity in these cells (Fig. 2,AC). Furthermore the absence of alkaline phosphatase activity in 2T3 cells expressing a dominant-negative PI 3-kinase conclusively establishes the requirement of PI 3-kinase for this osteoblast differentiation-specific early enzyme activity (Fig. 2,D and E). In addition, in long term culture, incubation of 2T3 cells with PI 3-kinase inhibitor Ly-294002 in the presence of BMP-2 completely blocked mineralized bone nodule formation indicating further the importance of this lipid kinase in osteoblast differentiation (data not shown).
      Many biological functions of PI 3-kinase are regulated by its direct downstream target, Akt serine/threonine kinase (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ,
      • Ghosh
      • Choudhury G.
      ,
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ). The products of PI 3-kinase, the D3-phosphoinositides, bind to the N-terminal plextrin homology (PH) domain of Akt to recruit it into the plasma membrane where PDK1 and PDK2 phosphorylate it, resulting in its full activation (
      • Toker A.
      • Newton A.C.
      ,
      • Franck T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ). Although a diverse group of proteins that function in many different cellular processes has been identified as substrates of Akt, the role of Akt in inhibition of apoptosis has been most extensively studied (
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ). This action of Akt is mediated by phosphorylation of pro-apoptotic protein BAD and caspase 9 (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S., Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ). Terminal cellular differentiation is a post-mitotic phenomenon where cells acquire an apoptosis-resistant phenotype (
      • Wang J.
      • Walsh K.
      ). Recently Akt has been implicated in myogenic differentiation (
      • Fujio Y.
      • Guo K.
      • Mano T.
      • Mitsuuchi Y.
      • Testa J.R.
      • Walsh K.
      ,
      • Jiang B-H.
      • Aoki M.
      • Zheng J.Z., Li, J.
      • Vogt P.K.
      ). Also Akt regulates IGF-induced myotube formation, as a downstream target of PI 3-kinase. This action of Akt is mediated by phosphorylation of Raf-1, which negatively regulates its kinase activity and results in maintenance of highly differentiated myotubes (
      • Rommel C.
      • Clark B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ). Here we show that the osteogenic factor BMP-2 stimulates Akt activity in a PI 3-kinase-dependent manner in 2T3 osteoblast precursor cells in response to BMP-2 (Fig. 3). Expression of dominant-negative Akt resulted in significant inhibition of BMP-2-induced alkaline phosphatase activity in these cells, indicating that Akt regulates the expression of this early osteoblast-specific marker protein (Fig. 4). Along with 2T3 cells, which we established and used extensively as a model for osteoblast growth and differentiation (
      • Ghosh-Choudhury N.
      • Harris M.A.
      • Feng J.Q.
      • Mundy G.R.
      • Harris S.E.
      ,
      • Ghosh-Choudhury N.
      • Windle J.J.
      • Koop B.A.
      • Harris M.A.
      • Guerrero D.L.
      • Mundy G.R.
      • Harris S.E.
      ,
      • Chen D., Ji X.
      • Harris M.A.
      • Feng J.Q.
      • Karsenty G.
      • Celeste A.J.
      • Rosen V.
      • Mundy G.R.
      • Harris S.E.
      ,
      • Mundy G.
      • Garrett R.
      • Harris S.
      • Chan J.
      • Chen D.
      • Rossini G.
      • Boyce B.
      • Zhao M.
      • Gutierrez G.
      ,
      • Yoshida Y.
      • Tanaka S.
      • Umemori H.
      • Minowa O.
      • Usui M.
      • Ikematsu N.
      • Hosoda E.
      • Yamashita T.
      • Miyazono K.
      • Noda M.
      • Yamamoto T.
      ), we used another multipotent cell line, C2C12, which has the potential to undergo osteoblast differentiation in the presence of BMP-2 (
      • Katagiri T.
      • Yamaguchi A.
      • Komaki M.
      • Abe E.
      • Takahashi N.
      • Ikeda t.
      • Rosen V.
      • Wozney J.M.
      • Fujisawa-Sehara A.
      • Suda T.
      ,
      • Akiyama S.
      • Katagiri T.
      • Namiki M.
      • Yamaji N.
      • Yamamoto N.
      • Miyama K.
      • Shibuya H.
      • Ueno N.
      • Wozney J.
      • Suda T.
      ). Similar to 2T3 cells, BMP-2 stimulated Akt kinase activity in these cells, and expression of dominant-negative Akt significantly blocked BMP-2-induced alkaline phosphatase activity (data not shown). These results further confirm our observation that PI 3-kinase/Akt signaling cascade regulates osteoblast differentiation in cells, which have the potential to undergo differentiation to osteoblasts in response to BMP-2.
      BMP-regulated Smad1 or Smad5 after dimerization with Smad4 binds to specific DNA elements present in the target genes (,
      • Massague J.
      ). Smads also interact with other transcription factors, transcriptional coactivators, and cell-specific DNA-binding proteins to specify transcriptional programs that determine the biological functions of BMPs (,
      • Massague J.
      ,
      • Hata A.
      • Seoane J.
      • Lagna G.
      • Montalvo E.
      • Hemmati-Brivaniou A.
      • Massague J.
      ). C-terminal phosphorylation of Smad by the type I BMP receptor is sufficient for its translocation to the nucleus, DNA binding, and stimulation of transcription (,
      • Massague J.
      ,
      • Heldin C.-H.
      • Myazono K.
      • ten Dijke P.
      ,
      • Liu F.
      • Hata A.
      • Baker J.C.
      • Doody J.
      • Carcamo J.
      • Harland R.M.
      • Massague J.
      ). Here we demonstrate that Smad5 and Smad1 stimulate BMP-2 transcription in 2T3 and C2C12 progenitor cells (Fig. 6 and data not shown).
      Expression of Smad proteins has been shown to induce transcription of target gene in a ligand-independent manner and partially mimics the action of the ligands (
      • Yang X.
      • Letterio J.J.
      • Lechleider J.L.
      • Chen L.
      • Hayman R., Gu, H.
      • Roberts A.B.
      • Deng C.
      ,
      • Piek E., Ju, W.J.
      • Heyer J.
      • Escalante-Alcalde D.
      • Stewart C.L.
      • Weinstein M.
      • Deng C.
      • Kucherlapati R.
      • Bottinger E.P.
      • Roberts A.B.
      ). However, the precise mechanism by which different signaling inputs regulate Smad-dependent transcription has not been extensively studied. For example ERK1/2-dependent serine phosphorylation of Smad1 in the linker domain has been shown to block its translocation to the nucleus and transcriptional activity, indicating a negative regulation of this transcription factor by this kinase (
      • Kretzschmar M.
      • Doody J.
      • Massague J.
      ). We have provided evidence here that the PI 3-kinase/Akt signaling cascade regulates Smad5-dependent BMP-2 transcription (Fig. 6). Although BMP-specific Smad5 and Smad1 do not contain any Akt phosphorylation site, we demonstrate that Akt regulates translocation of Smad5 and Smad1 into the nucleus (Fig. 7). Because Smad-dependent transcription is dependent upon its ability to be localized in the nucleus (
      • Liu F.
      • Hata A.
      • Baker J.C.
      • Doody J.
      • Carcamo J.
      • Harland R.M.
      • Massague J.
      ), our observation that Akt regulates Smad translocation provides a mechanism by which Akt modulates BMP-2-stimulated Smad-dependent transcription. These results represent the first demonstration of a signal transduction pathway that positively regulates Smad-dependent transcription of BMP-2gene. Furthermore, our data indicate that interplay between the PI 3-kinase/Akt signaling cascade and BMP-specific Smad-induced transcription of lineage-specific genes regulates osteoblastogenesis.

      Acknowledgments

      We thank Drs. Dan Riley and Jeff Barnes for critically reading the manuscript. We also thank Drs. Thomas Franke, Wataru Ogawa, and Joan Massague for providing us the plasmid constructs. We thank Dr. Anita Roberts for kindly providing us the adenovirus vectors expressing constitutively active BMPRIA and BMPRIB.

      REFERENCES

        • Reddi A.H.
        Curr. Opin. Genet. Dev. 1994; 4: 737-744
        • Sakou T.
        Bone. 1998; 22: 591-603
        • Ghosh-Choudhury N.
        • Harris M.A.
        • Feng J.Q.
        • Mundy G.R.
        • Harris S.E.
        Crit. Rev. Euk. Gene Exp. 1994; 4: 345-355
        • Wrana J.L.
        Cell. 2000; 100: 189-192
        • Massague J.
        Mol. Cell. Biol. 2000; 1: 169-178
        • Kawabata M.
        • Imamura T.
        • Miyaxono K.
        Cytokine Growth Factor Rev. 1998; 9: 49-61
        • Heldin C.-H.
        • Myazono K.
        • ten Dijke P.
        Nature. 1997; 390: 465-471
        • Karsenty G.
        Cell. Dev. Biol. 2000; 11: 343-346
        • Ducy P.
        • Schinke T.
        • Karcenty G.
        Science. 2000; 289: 1501-1504
        • Bonni A.
        • Sun Y.
        • Nadal-Vicens M.
        • Bhatt A.
        • Frank D.A.
        • Rozovsky I.
        • Stahl N.
        • Yancopoulos G.D.
        • Greenberg M.E.
        Science. 1997; 278: 477-483
        • Dudek H.
        • Datta S.R.
        • Franke T.F.
        • Birnbaum M.J.
        • Yao R.
        • Cooper G.M.
        • Segal R.A.
        • Kaplan D.R.
        • Greenberg M.E.
        Science. 1997; 275: 661-665
        • Fukuda M.
        • Gotoh Y.
        • Tachibana T.
        • Dell K.
        • Hattori S.
        • Yoneda Y.
        • Nishida E.
        Oncogene. 1995; 11: 239-244
        • Jiang B-H.
        • Zheng J.Z.
        • Vogt P.K.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14179-14183
        • Sakaue H.
        • Ogawa W.
        • Matsumoto M.
        • Kuroda S.
        • Takata M.
        • Suginoto T.
        • Spiegelman B.M.
        • Kasuga M.
        J. Biol. Chem. 1998; 273: 28945-28952
        • Shepherd P.R.
        • Withers D.J.
        • Siddle K.
        Biochem. J. 1998; 333: 471-490
        • Franke T.F.
        • Kaplan D.R.
        • Cantley L.C.
        Cell. 1997; 88: 435-437
        • Toker A.
        • Cantley L.C.
        Nature. 1997; 387: 673-676
        • Franke T.F.
        • Cantley L.C.
        Nature. 1997; 390: 116-117
        • Bakin A.V.
        • Tomlinson A.K.
        • Bhowmick N.A.
        • Moses H.L.
        • Arteaga C.L.
        J. Biol. Chem. 2000; 275: 36803-36810
        • Ghosh-Choudhury N.
        • Windle J.J.
        • Koop B.A.
        • Harris M.A.
        • Guerrero D.L.
        • Mundy G.R.
        • Harris S.E.
        Endocrinology. 1996; 137: 331-339
        • Chen D., Ji X.
        • Harris M.A.
        • Feng J.Q.
        • Karsenty G.
        • Celeste A.J.
        • Rosen V.
        • Mundy G.R.
        • Harris S.E.
        J. Cell Biol. 1998; 142: 295-305
        • Mundy G.
        • Garrett R.
        • Harris S.
        • Chan J.
        • Chen D.
        • Rossini G.
        • Boyce B.
        • Zhao M.
        • Gutierrez G.
        Science. 1999; 286: 1946-1949
        • Yoshida Y.
        • Tanaka S.
        • Umemori H.
        • Minowa O.
        • Usui M.
        • Ikematsu N.
        • Hosoda E.
        • Yamashita T.
        • Miyazono K.
        • Noda M.
        • Yamamoto T.
        Cell. 2000; 103: 1085-1097
        • Katagiri T.
        • Yamaguchi A.
        • Komaki M.
        • Abe E.
        • Takahashi N.
        • Ikeda t.
        • Rosen V.
        • Wozney J.M.
        • Fujisawa-Sehara A.
        • Suda T.
        J. Cell Biol. 1994; 127: 1755-1766
        • Ghosh
        • Choudhury G.
        J. Biol. Chem. 2001; 276: 35636-35643
        • Ghosh
        • Choudhury G.
        • Grandaliano G.
        • Jin D.-C.
        • Katz M.S.
        • Abboud H.E.
        Kidney Int. 2000; 57: 908-917
        • Ghosh Choudhury G.
        • Ricono J.M.
        Biochem. Biophys. Res. Commun. 2000; 273: 1069-1077
        • Ghosh-Choudhury N.
        • Ghosh Choudhury G.
        • Celeste A.
        • Ghosh P.M.
        • Moyer M.
        • Abboud S.L.
        • Kreisberg J.
        Biochim. Biophys. Acta. 2000; 1497: 186-196
        • Ghosh-Choudhury N.
        • Woodruff K., Qi W.
        • Celeste A.
        • Abboud S.L.
        • Ghosh Choudhury G.
        Biochem. Biophys. Res. Commun. 2000; 272: 705-711
        • Ghosh Choudhury G.
        • Kim Y.-S.
        • Simon M.
        • Wozney J.
        • Harris S.
        • Ghosh-Choudhury N.
        • Abboud H.E.
        J. Biol. Chem. 1999; 274: 10897-10902
        • Ghosh Choudhury G.
        • Jin D.-C.
        • Kim Y.-S.
        • Celeste A.
        • Ghosh-Choudhury N.
        • Abboud H.E.
        Biochem. Biophys. Res. Commun. 1999; 258: 490-496
        • Ghosh-Choudhury N.
        • Harris M.A.
        • Wozney J.
        • Mundy G.R.
        • Harris S.E.
        Biochem. Biophys. Res. Commun. 1997; 231: 196-202
        • Ghosh-Choudhury N.
        • Ghosh
        • Choudhury G.
        • Harris M.A.
        • Wozney J.
        • Mundy G.R.
        • Abboud S.L.
        • Harris S.E.
        Biochem. Biophys. Res. Commun. 2001; 286: 101-108
        • Quon M.J.
        • Chen H.
        • Ing B.L.
        • Liu M-L.
        • Zarnowski M.J.
        • Yonezawa K.
        • Kasuga M.
        • Cushman S.W.
        • Taylor S.I.
        Mol. Cell. Biol. 1995; 15: 5403-5411
        • Datta S.R.
        • Brunet A.
        • Greenberg M.E.
        Genes Dev. 1999; 13: 2905-2927
        • Yano S.
        • Tokumitsu H.
        • Soderling T.R.
        Nature. 1998; 396: 584-587
        • Toker A.
        • Newton A.C.
        Cell. 2000; 103: 185-188
        • Hoodless P.A.
        • Haerry T.
        • Abdollah S.
        • Stapleton M.
        • O'Connor M.B.
        • Attisano L.
        • Wrana J.L.
        Cell. 1996; 85: 489-500
        • Zou H.
        • Wieser R.
        • Massague J.
        • Niswander L.
        Genes Dev. 1997; 11: 2191-2203
        • Panchision D.M.
        • Pickel J.M.
        • Studerr L.
        • Lee S-H.
        • Turner P.A.
        • Hazel T.G.
        • McKay R.D.G.
        Genes Dev. 2001; 15: 2094-2110
        • Fujii M.
        • Takeda K.
        • Imamura T.
        • Aoki H.
        • Sampath T.K.
        • Enomoto S.
        • Kawabata M.
        • Kato M.
        • Ichijo H.
        • Miyazono K.
        Mol. Biol. Cell. 1999; 10: 3891-3913
        • Dike P.T.
        • Yamashita H.
        • Sampath T.K.
        • Reddi A.H.
        • Estevez M.
        • Riddle D.L.
        • Ichijo H.
        • Heldin C-H.
        • Miyazono K.
        J. Biol. Chem. 1994; 269: 16985-16988
        • Liu F.
        • Hata A.
        • Baker J.C.
        • Doody J.
        • Carcamo J.
        • Harland R.M.
        • Massague J.
        Nature. 1996; 381: 620-623
        • Shin I.
        • Bakin A.V.
        • Rodeck U.
        • Brunet A.
        • Arteaga C.L.
        Mol. Biol. Cell. 2001; 12: 3328-3339
        • Vinals F.
        • Pouyssegur J.
        Mol. Cell. Biol. 2001; 21: 7218-7230
        • Kaliman P.
        • Vinals F.
        • Testar X.
        • Palacin M.
        • Zorzano A.
        J. Biol. Chem. 1996; 271: 19146-19151
        • Pinset C.
        • Garcia A.
        • Rousse S.
        • Dubois C.
        • Montarras D.
        C. R. Acad Sci Ser. III. 1997; 320: 367-374
        • Kulik G.
        • Klippel A.
        • Weber M.J.
        Mol. Cell. Biol. 1997; 17: 1595-1606
        • Yao R.
        • Cooper G.M.
        Science. 1995; 267: 2003-2006
        • Franck T.F.
        • Kaplan D.R.
        • Cantley L.C.
        • Toker A.
        Science. 1997; 275: 665-668
        • Datta S.R.
        • Dudek H.
        • Tao X.
        • Masters S., Fu H.
        • Gotoh Y.
        • Greenberg M.E.
        Cell. 1997; 91: 231-241
        • Cardone M.H.
        • Roy N.
        • Stennicke H.R.
        • Salvesen G.S.
        • Franke T.F.
        • Stanbridge E.
        • Frisch S.
        • Reed J.C.
        Science. 1998; 282: 318-321
        • Wang J.
        • Walsh K.
        Science. 1996; 273: 359-361
        • Fujio Y.
        • Guo K.
        • Mano T.
        • Mitsuuchi Y.
        • Testa J.R.
        • Walsh K.
        Mol. Cell. Biol. 1999; 19: 5073-5082
        • Jiang B-H.
        • Aoki M.
        • Zheng J.Z., Li, J.
        • Vogt P.K.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2077-2081
        • Rommel C.
        • Clark B.A.
        • Zimmermann S.
        • Nunez L.
        • Rossman R.
        • Reid K.
        • Moelling K.
        • Yancopoulos G.D.
        • Glass D.J.
        Science. 1999; 286: 1738-1741
        • Akiyama S.
        • Katagiri T.
        • Namiki M.
        • Yamaji N.
        • Yamamoto N.
        • Miyama K.
        • Shibuya H.
        • Ueno N.
        • Wozney J.
        • Suda T.
        Exp. Cell Res. 1997; 235: 362-369
        • Hata A.
        • Seoane J.
        • Lagna G.
        • Montalvo E.
        • Hemmati-Brivaniou A.
        • Massague J.
        Cell. 2000; 100: 229-240
        • Yang X.
        • Letterio J.J.
        • Lechleider J.L.
        • Chen L.
        • Hayman R., Gu, H.
        • Roberts A.B.
        • Deng C.
        EMBO J. 1999; 18: 1280-1291
        • Piek E., Ju, W.J.
        • Heyer J.
        • Escalante-Alcalde D.
        • Stewart C.L.
        • Weinstein M.
        • Deng C.
        • Kucherlapati R.
        • Bottinger E.P.
        • Roberts A.B.
        J. Biol. Chem. 2001; 276: 19945-19953
        • Kretzschmar M.
        • Doody J.
        • Massague J.
        Nature. 1997; 389: 618-622

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