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Insulin-like Growth Factor-II, Phosphatidylinositol 3-Kinase, Nuclear Factor-κB and Inducible Nitric-oxide Synthase Define a Common Myogenic Signaling Pathway*

  • Perla Kaliman
    Correspondence
    To whom correspondence should be addressed. Tel.: 34-3-4021547; Fax: 34-3-4021559
    Footnotes
    Affiliations
    From the Departament de Bioquı́mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain
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  • Judith Canicio
    Footnotes
    Affiliations
    From the Departament de Bioquı́mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain
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  • Xavier Testar
    Affiliations
    From the Departament de Bioquı́mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain
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  • Manuel Palacı́n
    Affiliations
    From the Departament de Bioquı́mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain
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  • Antonio Zorzano
    Affiliations
    From the Departament de Bioquı́mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain
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  • Author Footnotes
    * This work was supported by Dirección General de Investigación Cientı́fica y Técnica Research Grant PB95/0971, Fondo de Investigación Sanitaria Research Grant 97/2101, and Generalitat de Catalunya Research Grants 1995 SGR-537 and 1997 SGR-121.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.
    ‡ These authors contributed equally to this work.
Open AccessPublished:June 18, 1999DOI:https://doi.org/10.1074/jbc.274.25.17437
      Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-κB (NF-κB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-κB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-κB inhibition. Both NF-κB and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-κB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substratel-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-κB, PI 3-kinase inhibition prevented NF-κB activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in IκB-α protein content that correlated with a decrease in the amount of IκB-α associated with p65 NF-κB.
      Skeletal muscle cell differentiation is a highly ordered multistep process that involves the expression of myogenic transcription factors, followed by cyclin kinase inhibitor p21 protein induction, cell cycle arrest, muscle-specific protein expression, and cell fusion to form multinucleated myotubes (
      • Andrés V.
      • Walsh K.
      ,
      • Guo K.
      • Wang J.
      • Andrés V.
      • Smith R.C.
      • Walsh K.
      ,
      • Olson E.N.
      ,
      • Weintraub H.
      ). The commitment to differentiate into myotubes is influenced negatively by several factors. Treatment of myoblasts with fetal bovine serum, basic fibroblast growth factor 2, or transforming growth factor β1 is known to inhibit differentiation of myoblasts (
      • Brennan T.J.
      • Edmondson D.G.
      • Li L.
      • Olson E.N.
      ,
      • Li L.
      • Zhou J.
      • James G.
      • Heller-Harrison R.
      • Czech M.P.
      • Olson E.N.
      ). Myogenesis is also regulated negatively by oncogenes such as c-fos, Ha-ras, and E1a (
      • Caruso M.
      • Martelli F.
      • Giordano A.
      • Felsani A.
      ,
      • Kong Y.
      • Johnson S.E.
      • Taparowsky E.J.
      • Konieczny S.F.
      ,
      • Lassar A.B.
      • Thayer M.J.
      • Overell R.W.
      • Weintraub H.
      ). The insulin-like growth factors (IGFs)
      The abbreviations used are: IGF, insulin-like growth factor; DMEM, Dulbecco's modified Eagle's medium; PI, phosphatidylinositol; NOS, nitric-oxide synthase; iNOS, inducible NOS; cNOS, constitutive NOS; eNOS, endothelial NOS; NaSal, sodium salicylate; NNA, N ω-nitro-l-arginine; l-Arg, l-arginine; l-Lys, l-lysine; PDTC, pyrrolidinedithiocarbamic acid; NF-κB, nuclear factor κB; PBS, phosphate-buffered saline.
      1The abbreviations used are: IGF, insulin-like growth factor; DMEM, Dulbecco's modified Eagle's medium; PI, phosphatidylinositol; NOS, nitric-oxide synthase; iNOS, inducible NOS; cNOS, constitutive NOS; eNOS, endothelial NOS; NaSal, sodium salicylate; NNA, N ω-nitro-l-arginine; l-Arg, l-arginine; l-Lys, l-lysine; PDTC, pyrrolidinedithiocarbamic acid; NF-κB, nuclear factor κB; PBS, phosphate-buffered saline.
      are the only known growth factors that are crucial to myogenesis (
      • Florini J.R.
      • Ewton D.Z.
      • Magri K.A.
      ,
      • Florini J.R.
      • Ewton D.Z.
      • Coolican S.A.
      ). IGF expression is increased during myoblast differentiation in response to serum withdrawal (
      • Kou K.
      • Rotwein P.S.
      ,
      • Rosen K.M.
      • Wentworth B.M.
      • Rosenthal N.
      • Villa-Komaroff L.
      ,
      • Tollefsen S.
      • Lajara R.
      • McCusker R.H.
      • Clemmons D.R.
      • Rotwein P.
      ,
      • Tollefsen S.
      • Levis Sadow J.
      • Rotwein P.
      ). The amount of IGF-II secreted correlates with the rate of spontaneous differentiation that, in the absence of exogenous IGF-II, can be inhibited by antisense oligonucleotides complementary to IGF-II mRNA (
      • Florini J.R.
      • Magri K.A.
      • Ewton D.Z.
      • James P.L.
      • Grindstaff K.
      • Rotwein P.S.
      ). Because of their myogenic actions, IGFs have been postulated as potential therapeutic tools in conditions characterized by muscle myopathy, atrophy, or muscle injury. In this context, IGFs have been implicated in the regulation of satellite cell function during regeneration, a characteristic response of adult muscle to injury (
      • Edwall D.
      • Schalling M.
      • Jennische E.
      • Norstedt G.
      ,
      • Levinovitz A.
      • Jennische E.
      • Oldfors A.
      • Edwall D.
      • Norstedt G.
      ). However, IGFs are pleiotropic growth factors, and they also affect the growth of several tissues other than skeletal muscle. This has led to the analysis of the intracellular myogenic process initiated by IGFs. It is known that IGF-I and IGF-II switch on the myogenic program by activating the IGF-I receptor (
      • Ewton D.Z.
      • Falen S.L.
      • Florini J.R.
      ). During the last 2 years, the phosphatidylinositol (PI) 3-kinase has emerged as an essential second messenger for skeletal muscle cell differentiation (
      • Kaliman P.
      • Zorzano A.
      ,
      • Kaliman P.
      • Viñals F.
      • Testar X.
      • Palacı́n M.
      • Zorzano A.
      ,
      • Coolican S.A.
      • Samuel D.S.
      • Ewton D.Z.
      • McWade F.J.
      • Florini J.R.
      ). Moreover, by overexpressing a mutant p85 regulatory subunit of PI 3-kinase (Δp85) lacking the ability to bind and activate the p110 catalytic subunit (L6E9-Δp85), we showed that the heterodimeric p85-p110 is the PI 3-kinase isoform essential for IGF-induced myogenesis in L6E9 muscle cells (
      • Kaliman P.
      • Canicio J.
      • Shepherd P.R.
      • Beeton C.A.
      • Testar X.
      • Palacı́n M.
      • Zorzano A.
      ). Currently, there is no information regarding the downstream signals activated by IGFs and PI 3-kinase or its PI 3-phosphate products during myogenesis, and we have recently shown that the serine/threonine p70 S6 kinase, a downstream element in several PI 3-kinase-dependent signaling cascades (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ), is not involved in the myogenic actions of IGFs in rat, mouse, or human cells (
      • Canicio J.
      • Gallardo E.
      • Illa I.
      • Testar X.
      • Palacı́n M.
      • Zorzano A.
      • Kaliman P.
      ).
      Among the signaling events involved in myogenesis, the induction of chick embryonic myoblast fusion in low serum conditions requires NO production and NF-κB activity (
      • Lee K.H.
      • Baek M.Y.
      • Moon K.Y.
      • Song W.K.
      • Chung C.H.
      • Ha D.B.
      • Kang M.-S.
      ,
      • Lee K.H.
      • Kim D.G.
      • Shin N.Y.
      • Song W.K.
      • Kwon H.
      • Chung C.H.
      • Kang M.-S.
      ). However, the mechanisms that trigger NF-κB and NOS activation in differentiating myoblasts and the involvement of these molecules in biochemical differentiation (i.e. expression of structural and functional muscle markers) remain to be defined. In the present study, we attempted to further characterize the myogenic intracellular pathway depending on IGF-II and PI 3-kinase. We describe here a myogenic signaling cascade initiated by IGF-II that leads to biochemical and morphological skeletal muscle cell differentiation and that involves (i) PI 3-kinase activation, (ii) IκB-α degradation and dissociation from p65 NF-κB, (iii) NF-κB activation, and (iv) iNOS expression and activation. Moreover, we show the ability of the NOS substratel-Arg to induce myogenesis in the absence of IGFs in both rat and human skeletal muscle cells.

      DISCUSSION

      Results presented here define a model for the IGF-II-induced myogenic pathway, in which PI 3-kinase, NF-κB, and NOS activities are critical elements. Our data show that IGF-II-induced myogenesis can be blocked by either PI 3-kinase inhibitors, NF-κB inhibitors, or NOS inhibitors. Several lines of evidence indicate that PI 3-kinase, NF-κB, and iNOS are elements of a common myogenic cascade: (i) IGF-II-induced NF-κB activation was blocked by PI 3-kinase inhibition, (ii) IGF-II-induced iNOS expression correlated in time with the activation of NF-κB and was blocked by either PI 3-kinase or NF-κB inhibitors, and (iii) IGF-II-induced iNOS expression was accompanied by an IGF-II-induced NO production that was blocked by PI 3-kinase, NF-κB, or NOS inhibitors.
      An early event in the IGF-II-induced differentiation program was the increase in IκB-α and the p65 NF-κB subunit protein content. These data are consistent with the evidence that NF-κB and IκB are components of a mutual regulatory system in which the levels of one component control the activity or quantity of the other (
      • Baldwin Jr., A.S.
      ). After 24 h of IGF-II treatment, a transient peak of NF-κB DNA binding activity was observed that correlated with a decrease in IκB-α protein levels. It has been reported that differentiation of skeletal muscle cells in a medium containing low serum concentration induced a down-regulation of the activity of NF-κB and other proliferating transcription factors (
      • Lehtinen S.K.
      • Rahkila P.
      • Helenius M.
      • Korhonen P.
      • Salminen A.
      ). The authors did not consider the effects of IGFs in their systems, and they established from their data that there was a causative relation between NF-κB down-regulation and myogenesis. However, the authors did not examine whether the NF-κB activity detected after 24 h in a low serum differentiation medium, even if it was lower than that observed during proliferation, was necessary for differentiation. Indeed, this appears to be the case, because consistent with our observations for rat and human myoblasts differentiated with IGF-II, Lee et al. (
      • Lee K.H.
      • Kim D.G.
      • Shin N.Y.
      • Song W.K.
      • Kwon H.
      • Chung C.H.
      • Kang M.-S.
      ) reported that chick embryonic myoblasts require NF-κB activity to fuse in a low serum-containing differentiation medium.
      The effect of IGF-II in modulating NF-κB activity in L6E9 myoblasts appears to be tightly regulated by PI 3-kinase activity. Our results indicate that PI 3-kinase is required for NF-κB activation during myoblast differentiation through a mechanism that involves IκB-α degradation. The peak of NF-κB DNA binding activity after 24 h of IGF-II treatment was blocked by PI 3-kinase inhibition, and this correlated in time with higher amounts of IκB-α protein and higher amounts of inactive p65 NF-κB complexed with IκB-α in the presence of LY294002 than those detected in control myoblasts.
      Our results suggest a biphasic effect of IGF-II in modulating NF-κB activity in L6E9 myoblasts: during the first 12 h, IGF-II induced the up-regulation of IκB-α and p65 expression in a PI 3-kinase-independent way, and from 12 to 24 h, IGF-II induced the dissociation of IκB-α from p65 and the degradation of IκB-α through PI 3-kinase-dependent mechanisms. We interpret the complexity of these results as an example of the diversity of signals that can be elicited when a growth factor activates a tyrosine kinase receptor and generates a set of distinct second messengers in the cell. In this particular case, the expression of p65 and IkB-α is induced by IGF-II through yet undefined signaling pathways that do not involve PI 3-kinase activity. In contrast, the degradation of IkB-α and its dissociation from p65 require PI 3-kinase activity. In this latter case, it is tempting to hypothesize that PI 3-kinase could be acting upstream from the IkB kinases responsible for NF-κB activation. Indeed, many if not all activators of NF-κB have been reported to induce degradation of IκB-α (
      • Beg A.A.
      • Ruben S.M.
      • Scheinman R.I.
      • Haskill S.
      • Rosen C.A.
      • Baldwin Jr., A.S.
      ,
      • Henkel T.
      • Machleidt T.
      • Alkalay I.
      • Krönke M.
      • Ben-Neriah Y.
      • Baeuerle P.A.
      ) by activating IκB kinases that phosphorylate IκB-α on serines 32 and 36 within the N-terminal regulatory domain (
      • DiDonato J.A.
      • Hayakawa M.
      • Rothwarf D.M.
      • Zandi E.
      • Karin M.
      ,
      • Mercurio F.
      • Zhu H.
      • Murray B.W.
      • Shevchenko A.
      • Bennett B.L.
      • Li J.
      • Young D.B.
      • Barbosa M.
      • Mann M.
      • Manning A.
      • Rao A.
      ,
      • Regnier C.H.
      • Song H.Y.
      • Gao X.
      • Goeddel D.V.
      • Cao Z.
      • Rothe M.
      ,
      • Woronicz J.D.
      • Gao X.
      • Cao Z.
      • Rothe M.
      • Goeddel D.V.
      ,
      • Zandi E.
      • Rothwarf D.M.
      • Delhase M.
      • Hayakawa M.
      • Karin M.
      ). This is the most probable mechanism by which IGF-II and PI 3-kinase induce NF-κB activation in differentiating myoblasts, because we did not detect any IGF-II-induced IκB-α tyrosine phosphorylation, which represents a proteolysis-independent mechanism for NF-κB activation (data not shown) (
      • Imbert V.
      • Rupec R.A.
      • Livolsi A.
      • Pahl H.L.
      • Traeckner E.B.M.
      • Mueller-Dieckmann C.
      • Farahifar D.
      • Rossi B.
      • Auberger P.
      • Baeuerle P.A.
      • Peyron J.-F.
      ). Potential kinases acting between PI 3-kinase and IkB kinases during IGF-II myogenesis could be the protein kinase B and some PKC isoforms that are activated by PI 3-kinase products (
      • Cross D.A.E.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ,
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ). In this context, ζPKC and εPKC have been implicated in NF-κB activation (
      • Dı́az-Guerra M.J.M.
      • Bodel-n O.G.
      • Velasco M.
      • Whelan R.
      • Parker P.
      • Boscá L.
      ,
      • Dı́az-Meco M.T.
      • Domı́nguez I.
      • Sanz L.
      • Dent P.
      • Lozano J.
      • Municio M.M.
      • Berra E.
      • Hay R.T.
      • Sturgill T.W.
      • Moscat J.
      ).
      We show here that iNOS expression and activation are early events during IGF-II-induced myogenesis. Moreover, we describe here the ability of the NOS substrate l-Arg to induce myogenesis in the absence of IGFs in both rat and human skeletal muscle cells. The identification of direct targets for NO action in myogenesis is a matter of current interest, and one gene that may be regulated by NO in skeletal muscle cells is the Cdk inhibitor p21. It has been reported that NO blocks the cell cycle progression at the G1/S transition by inhibiting Cdk2-mediated phosphorylation of the retinoblastoma protein and that the p21 induction is involved in the Cdk2 inhibition (
      • Ishida A.
      • Sasaguri T.
      • Kosaka C.
      • Nojima H.
      • Ogata J.
      ). A causative role for NO action in nerve growth factor-induced cell cycle exit and differentiation of PC12 neuronal cells has been reported (
      • Peunova N.
      • Enikolopov G.
      ). It has been proposed that NO diffusion from the producer cell could promote cessation of growth in adjacent cells, contributing to synchronization of development from precursor cells. The expression of Cdk inhibitor p21 and cell cycle exit are prerequisites for myogenesis, and these events, which are both induced by IGF-II in L6E9 cells, may be mediated by NO (
      • Andrés V.
      • Walsh K.
      ,
      • Canicio J.
      • Gallardo E.
      • Illa I.
      • Testar X.
      • Palacı́n M.
      • Zorzano A.
      • Kaliman P.
      ,
      • Kaliman P.
      • Canicio J.
      • Shepherd P.R.
      • Beeton C.A.
      • Testar X.
      • Palacı́n M.
      • Zorzano A.
      ). Results presented here suggest that IGF-II-dependent myoblast differentiation can be mimicked by l-Arg treatment, both at the biochemical and at the morphological level, and this ability ofl-Arg to induce myogenesis in the absence of IGFs may contribute to the development of new strategies for the treatment of myopathies.

      Acknowledgments

      We thank Robin Rycroft for editorial support and Dr. M. Camps, Dr. R. Casaroli, and S. Castel (Servei Cientı́fico Tècnics, University of Barcelona) for expert advice in microscopy techniques. We are grateful to Dr. I. Illa and E. Gallardo (Departament de Neurologia, Hospital Universitari de la Santa Creu i de Sant Pau, Barcelona) for providing human skeletal muscle biopsies and to Dr. J.-F. Peyron (Inserm U364, Nice, France) and Dr. T. Carbonell and Dr. Jeśus Ródenas (Departament de Fisiologia Animal, Universitat de Barcelona) for helpful discussions.

      REFERENCES

        • Andrés V.
        • Walsh K.
        J. Cell Biol. 1996; 132: 657-666
        • Guo K.
        • Wang J.
        • Andrés V.
        • Smith R.C.
        • Walsh K.
        Mol. Cell. Biol. 1995; 15: 3823-3829
        • Olson E.N.
        Dev. Biol. 1992; 154: 261-272
        • Weintraub H.
        Cell. 1993; 75: 1241-1244
        • Brennan T.J.
        • Edmondson D.G.
        • Li L.
        • Olson E.N.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3822-3826
        • Li L.
        • Zhou J.
        • James G.
        • Heller-Harrison R.
        • Czech M.P.
        • Olson E.N.
        Cell. 1992; 71: 1181-1194
        • Caruso M.
        • Martelli F.
        • Giordano A.
        • Felsani A.
        Oncogene. 1993; 8: 267-278
        • Kong Y.
        • Johnson S.E.
        • Taparowsky E.J.
        • Konieczny S.F.
        Mol. Cell. Biol. 1995; 15: 5205-5213
        • Lassar A.B.
        • Thayer M.J.
        • Overell R.W.
        • Weintraub H.
        Cell. 1989; 58: 659-667
        • Florini J.R.
        • Ewton D.Z.
        • Magri K.A.
        Annu. Rev. Physiol. 1991; 53: 201-216
        • Florini J.R.
        • Ewton D.Z.
        • Coolican S.A.
        Endocr. Rev. 1996; 17: 481-517
        • Kou K.
        • Rotwein P.S.
        Mol. Endocrinol. 1993; 7: 291-302
        • Rosen K.M.
        • Wentworth B.M.
        • Rosenthal N.
        • Villa-Komaroff L.
        Endocrinology. 1993; 133: 474-481
        • Tollefsen S.
        • Lajara R.
        • McCusker R.H.
        • Clemmons D.R.
        • Rotwein P.
        J. Biol. Chem. 1989; 264: 13810-13817
        • Tollefsen S.
        • Levis Sadow J.
        • Rotwein P.
        Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1543-1547
        • Florini J.R.
        • Magri K.A.
        • Ewton D.Z.
        • James P.L.
        • Grindstaff K.
        • Rotwein P.S.
        J. Biol. Chem. 1991; 266: 15917-15923
        • Edwall D.
        • Schalling M.
        • Jennische E.
        • Norstedt G.
        Endocrinology. 1989; 124: 820-825
        • Levinovitz A.
        • Jennische E.
        • Oldfors A.
        • Edwall D.
        • Norstedt G.
        Mol. Endocrinol. 1992; 6: 1227-1234
        • Ewton D.Z.
        • Falen S.L.
        • Florini J.R.
        Endocrinology. 1987; 120: 115-123
        • Kaliman P.
        • Zorzano A.
        Trends Cardiovasc. Med. 1997; 7: 198-202
        • Kaliman P.
        • Viñals F.
        • Testar X.
        • Palacı́n M.
        • Zorzano A.
        J. Biol. Chem. 1996; 271: 19146-19151
        • Coolican S.A.
        • Samuel D.S.
        • Ewton D.Z.
        • McWade F.J.
        • Florini J.R.
        J. Biol. Chem. 1997; 272: 6653-6662
        • Kaliman P.
        • Canicio J.
        • Shepherd P.R.
        • Beeton C.A.
        • Testar X.
        • Palacı́n M.
        • Zorzano A.
        Mol. Endocrinol. 1998; 12: 66-77
        • Cheatham B.
        • Vlahos C.J.
        • Cheatham L.
        • Wang L.
        • Blenis J.
        • Kahn C.R.
        Mol. Cell. Biol. 1994; 14: 4902-4911
        • Chung J.
        • Grammer T.C.
        • Lemon K.P.
        • Kazlauskas A.
        • Blenis J.
        Nature. 1994; 370: 71-75
        • Canicio J.
        • Gallardo E.
        • Illa I.
        • Testar X.
        • Palacı́n M.
        • Zorzano A.
        • Kaliman P.
        Endocrinology. 1998; 139: 5042-5049
        • Lee K.H.
        • Baek M.Y.
        • Moon K.Y.
        • Song W.K.
        • Chung C.H.
        • Ha D.B.
        • Kang M.-S.
        J. Biol. Chem. 1994; 269: 14371-14374
        • Lee K.H.
        • Kim D.G.
        • Shin N.Y.
        • Song W.K.
        • Kwon H.
        • Chung C.H.
        • Kang M.-S.
        Biochem. J. 1997; 324: 237-242
        • Vlahos C.J.
        • Matter W.F.
        • Hui K.Y.
        • Brown R.F.
        J. Biol. Chem. 1994; 269: 5241-5248
        • Castelló A.
        • Rodrı́guez-Manzaneque J.C.
        • Camps M.
        • Pérez-Castillo A.
        • Testar X.
        • Palacı́n M.
        • Santos A.
        • Zorzano A.
        J. Biol. Chem. 1994; 269: 5905-5912
        • Pujades C.
        • Forsberg E.
        • Enrich C.
        • Johansson S.
        J. Cell Sci. 1992; 102: 815-820
        • Laemmli U.K.
        Nature. 1970; 227: 680-685
        • Baeuerle P.
        • Baltimore D.
        Science. 1988; 242: 540-546
        • Kopp E.
        • Ghosh S.
        Science. 1994; 265: 956-959
        • Schreck R.
        • Meier B.
        • Männel D.N.
        • Dröge W.
        • Baeuerle P.A.
        J. Exp. Med. 1992; 175: 1181-1194
        • Brenman J.E.
        • Chao D.S.
        • Xia H.
        • Aldape K.
        • Bredt D.S.
        Cell. 1995; 82: 742-752
        • Kobzic L.
        • Stringer B.
        • Balligand J.L.
        • Reid M.B.
        • Stamler J.S.
        Biochem. Biophys. Res. Commun. 1995; 211: 375-381
        • Thanos D.
        • Maniatis T.
        Cell. 1995; 80: 529-532
        • Baldwin Jr., A.S.
        Annu. Rev. Immunol. 1996; 14: 649-681
        • Lehtinen S.K.
        • Rahkila P.
        • Helenius M.
        • Korhonen P.
        • Salminen A.
        Biochem. Biophys. Res. Com. 1996; 229: 36-43
        • Beg A.A.
        • Ruben S.M.
        • Scheinman R.I.
        • Haskill S.
        • Rosen C.A.
        • Baldwin Jr., A.S.
        Genes Dev. 1992; 6: 1899-1913
        • Henkel T.
        • Machleidt T.
        • Alkalay I.
        • Krönke M.
        • Ben-Neriah Y.
        • Baeuerle P.A.
        Nature. 1993; 365: 182-185
        • DiDonato J.A.
        • Hayakawa M.
        • Rothwarf D.M.
        • Zandi E.
        • Karin M.
        Nature. 1997; 388: 548-554
        • Mercurio F.
        • Zhu H.
        • Murray B.W.
        • Shevchenko A.
        • Bennett B.L.
        • Li J.
        • Young D.B.
        • Barbosa M.
        • Mann M.
        • Manning A.
        • Rao A.
        Science. 1997; 278: 860-866
        • Regnier C.H.
        • Song H.Y.
        • Gao X.
        • Goeddel D.V.
        • Cao Z.
        • Rothe M.
        Cell. 1997; 90: 373-383
        • Woronicz J.D.
        • Gao X.
        • Cao Z.
        • Rothe M.
        • Goeddel D.V.
        Science. 1997; 278: 866-869
        • Zandi E.
        • Rothwarf D.M.
        • Delhase M.
        • Hayakawa M.
        • Karin M.
        Cell. 1997; 91: 243-252
        • Imbert V.
        • Rupec R.A.
        • Livolsi A.
        • Pahl H.L.
        • Traeckner E.B.M.
        • Mueller-Dieckmann C.
        • Farahifar D.
        • Rossi B.
        • Auberger P.
        • Baeuerle P.A.
        • Peyron J.-F.
        Cell. 1996; 86: 787-798
        • Cross D.A.E.
        • Alessi D.R.
        • Cohen P.
        • Andjelkovich M.
        • Hemmings B.A.
        Nature. 1995; 378: 785-789
        • Nakanishi H.
        • Brewer K.A.
        • Exton J.H.
        J. Biol. Chem. 1993; 268: 13-16
        • Toker A.
        • Meyer M.
        • Reddy K.K.
        • Falck J.R.
        • Aneja R.
        • Aneja S.
        • Parra A.
        • Burns D.J.
        • Ballas L.M.
        • Cantley L.C.
        J. Biol. Chem. 1994; 269: 32358-32367
        • Dı́az-Guerra M.J.M.
        • Bodel-n O.G.
        • Velasco M.
        • Whelan R.
        • Parker P.
        • Boscá L.
        J. Biol. Chem. 1996; 271: 32028-32033
        • Dı́az-Meco M.T.
        • Domı́nguez I.
        • Sanz L.
        • Dent P.
        • Lozano J.
        • Municio M.M.
        • Berra E.
        • Hay R.T.
        • Sturgill T.W.
        • Moscat J.
        EMBO J. 1994; 13: 2842-2848
        • Ishida A.
        • Sasaguri T.
        • Kosaka C.
        • Nojima H.
        • Ogata J.
        J. Biol. Chem. 1997; 272: 10050-10057
        • Peunova N.
        • Enikolopov G.
        Nature. 1995; 375: 68-73