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Protein Kinase C-ζ and Phosphoinositide-dependent Protein Kinase-1 Are Required for Insulin-induced Activation of ERK in Rat Adipocytes

1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês

10.1074/jbc.274.43.30495

1083-351X

Mini P. Sajan, Mary L. Standaert, Gautam Bandyopadhyay, Michael J. Quon, Terrence R. Burke, Robert V. Farese,

PI3K/AKT/mTOR signaling in cancer

The mechanisms used by insulin to activate the multifunctional intracellular effectors, extracellular signal-regulated kinases 1 and 2 (ERK1/2), are only partly understood and appear to vary in different cell types. Presently, in rat adipocytes, we found that insulin-induced activation of ERK was blocked (a) by chemical inhibitors of both phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC)-ζ, and, moreover, (b) by transient expression of both dominant-negative Δp85 PI3K subunit and kinase-inactive PKC-ζ. Further, insulin effects on ERK were inhibited by kinase-inactive 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and by mutation of Thr-410 in the activation loop of PKC-ζ, which is the target of PDK-1 and is essential for PI3K/PDK-1-dependent activation of PKC-ζ. In addition to requirements for PI3K, PDK-1, and PKC-ζ, we found that a tyrosine kinase (presumably the insulin receptor), the SH2 domain of GRB2, SOS, RAS, RAF, and MEK1 were required for insulin effects on ERK in the rat adipocyte. Our findings therefore suggested that PDK-1 and PKC-ζ serve as a downstream effectors of PI3K, and act in conjunction with GRB2, SOS, RAS, and RAF, to activate MEK and ERK during insulin action in rat adipocytes. The mechanisms used by insulin to activate the multifunctional intracellular effectors, extracellular signal-regulated kinases 1 and 2 (ERK1/2), are only partly understood and appear to vary in different cell types. Presently, in rat adipocytes, we found that insulin-induced activation of ERK was blocked (a) by chemical inhibitors of both phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC)-ζ, and, moreover, (b) by transient expression of both dominant-negative Δp85 PI3K subunit and kinase-inactive PKC-ζ. Further, insulin effects on ERK were inhibited by kinase-inactive 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and by mutation of Thr-410 in the activation loop of PKC-ζ, which is the target of PDK-1 and is essential for PI3K/PDK-1-dependent activation of PKC-ζ. In addition to requirements for PI3K, PDK-1, and PKC-ζ, we found that a tyrosine kinase (presumably the insulin receptor), the SH2 domain of GRB2, SOS, RAS, RAF, and MEK1 were required for insulin effects on ERK in the rat adipocyte. Our findings therefore suggested that PDK-1 and PKC-ζ serve as a downstream effectors of PI3K, and act in conjunction with GRB2, SOS, RAS, and RAF, to activate MEK and ERK during insulin action in rat adipocytes. extracellular signal-regulated kinase insulin receptor substrate phosphatidylinositol 3-kinase protein kinase C Krebs-Ringer phosphate hemagglutinin wild type kinase-inactive Src homology 2 3-phosphoinositide-dependent protein kinase-1 Mitogen-activated protein kinases, extracellular signal-regulated kinases (ERKs)1 1 and 2, are activated by insulin through a mechanism involving tyrosine phosphorylation of insulin receptor substrate (IRS) family members or SHC, followed by sequential activation of GRB2, SOS, RAS, RAF, and MEK, which phosphorylates threonine and tyrosine residues on ERK1/2 (1Denton R.M. Tavaré J.M. Eur. J. Biochem. 1995; 227: 597-611Crossref PubMed Scopus (130) Google Scholar). Although ERK1/2 activation may occur independently of phosphatidylinositol 3-kinase (PI3K) in some cell types (2Yamamoto-Honda R. Tobe K. Kaburagi Y. Ueki K. Asai S. Yachi M. Shirouzu M. Yodoi J. Akanuma Y. Yokoyama S. Yazaki Y. Kadowaki T. J. Biol. Chem. 1995; 270: 2729-2734Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), inhibitors of PI3K have been reported to inhibit insulin-induced increases in ERK1/2 activity in a number of important cell types, including L6 myotubes (3Cross D.A.E. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (420) Google Scholar), Chinese hamster ovary cells (4Welsh G.I. Foulstone E.J. Young S.W. Tavaré J.M. Proud C.G. Biochem. J. 1994; 303: 15-20Crossref PubMed Scopus (183) Google Scholar), rat adipocytes (5Standaert M.L. Bandyopadhyay G. Farese R.V. Biochem. Biophy. Res. Commun. 1995; 209: 1082-1088Crossref PubMed Scopus (42) Google Scholar), 3T3/L1 adipocytes (6Suga J. Yoshimasa Y. Yamada K. Yamamoto Y. Inoue G. Okamoto M. Hayashi T. Shigemoto M. Kosaki A. Kuzuya H. Nakao K. Diabetes. 1997; 46: 735-741Crossref PubMed Scopus (44) Google Scholar), rat brown fat cells (7Shimizu Y. Tanishita T. Minokoshi Y. Shimazu T. Endocrinology. 1997; 138: 248-253Crossref PubMed Scopus (41) Google Scholar), human hepatoma Hep3B cells (8Lin Y.L. Chou C.K. Biochem. Biophys. Res. Commun. 1998; 246: 172-175Crossref PubMed Scopus (11) Google Scholar), and rat hepatocytes (9Band C.J. Posner B.I. J. Biol. Chem. 1997; 272: 138-145Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). This apparent dependence of insulin-stimulated ERK1/2 activation on PI3K has been neither confirmed by other experimental approaches nor satisfactorily explained in relationship to other signaling factors. In this regard, PI3K has been suggested to function downstream (10Kodaki T. Woscholski R. Hallberg B. Rodriquez-Viciana P. Downward J. Parker P.J. Curr. Biol. 1994; 4: 798-806Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 11Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1726) Google Scholar) or upstream (12Hu Q. Klippe l A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (517) Google Scholar) of RAS, but insulin does not appear to activate PI3K via RAS (13Gnudi L. Frevert E.U. Houseknecht K.L. Erhardt P. Kahn B.B. Mol. Endocrinol. 1997; 11: 67-76Crossref PubMed Scopus (25) Google Scholar). Presently, in rat adipocytes, we confirmed that PI3K was required, along with GRB2, SOS, RAS, RAF, and MEK1, for insulin-induced activation of ERK2; moreover, we found that downstream effectors of PI3K, viz., 3-phosphoinositide-dependent protein kinase-1 (PDK-1) and protein kinase C (PKC)-ζ, were also required for insulin-induced activation of ERK2. As described (5Standaert M.L. Bandyopadhyay G. Farese R.V. Biochem. Biophy. Res. Commun. 1995; 209: 1082-1088Crossref PubMed Scopus (42) Google Scholar, 14Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 15Yang Y. Farese R.V. FEBS Lett. 1993; 333: 287-290Crossref PubMed Scopus (23) Google Scholar), adipocytes were isolated by collagenase digestion of epididymal fat pads of 250-g male Harlan Sprague-Dawley rats, and suspended in glucose-free Krebs-Ringer phosphate (KRP) medium containing 1% bovine serum albumin. In some experiments, where indicated, the cells were equilibrated with 100 nm wortmannin (Sigma), 100 μm LY294002 (Alexis), 10 μm PD098059 (Alexis), or 10 or 100 μm genistein (Calbiochem) for 15 min, or for 60 min with myristoylated PKC-ζ pseudosubstrate (see Ref. 14Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar) (Quality Controlled Biochemicals Inc., Hopkington, MA), or for 180 min with a GRB2 SH2 domain inhibitor, a phosphotyrosine pY mimetic, viz., compound l-20d, an Nα-oxalyl-tripeptide containing a (phosphonomethyl)phenylalanine residue (see Ref. 16Yao Z. Richter King C. Cao T. Kelly J. Milne G.W.A. Voight J.H. Burke Jr., T.R. J. Med. Chem. 1998; 42: 25-35Crossref Scopus (94) Google Scholar), and then treated with or without 10 nm insulin for 10 min (this time was optimal for observing changes in ERK). As described in previous studies (5Standaert M.L. Bandyopadhyay G. Farese R.V. Biochem. Biophy. Res. Commun. 1995; 209: 1082-1088Crossref PubMed Scopus (42) Google Scholar, 15Yang Y. Farese R.V. FEBS Lett. 1993; 333: 287-290Crossref PubMed Scopus (23) Google Scholar) of total mitogen-activated protein kinase activation, after incubation, adipocytes were sonicated in buffer containing 40 mm β-glycerophosphate (pH, 7.3), 0.5 mmdithiothreitol, 0.75 mm EGTA, 0.15 mmNa3VO4, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 0.1 mm phenylmethylsulfonyl fluoride, and 5 μg/ml trypsin inhibitor. The resulting homogenates were centrifuged at 700 × g for 10 min to remove fat, cell debris, and nuclei. Post-nuclear supernatants were then supplemented with 0.154m NaCl, 1% Triton X-100, and 0.5% Nonidet, and equal amounts of lysate protein in each experiment (varying from 200 to 500 μg between experiments) were subjected to overnight immunoprecipitation at 4 °C with mouse monoclonal anti-ERK2 antibodies (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA), which, as shown below, immunoprecipitated ERK1, as well as ERK2, despite the fact that these antibodies reacted only with ERK2 in Western analyses. Precipitates were collected on Protein-AG-agarose beads, washed and incubated for 10 min at 30 °C in 50 μl of buffer containing 25 mm β-glycerophosphate (pH, 7.3), 0.5 mmdithiothreitol, 1.25 mm EGTA, 0.5 mmNa3VO4, 10 mm MgCl2, 1 mg/ml bovine serum albumin, 1 μm okadaic acid, 0.1 mm [γ-32P]ATP (NEN Life Science Products; approximate specific activity, 1,500,000 dpm/nmol), and 50 μg of myelin basic protein (Sigma). After incubation, an aliquot of the reaction mixture was spotted on p81 filter paper, which was washed and counted for 32P-radioactivity (5Standaert M.L. Bandyopadhyay G. Farese R.V. Biochem. Biophy. Res. Commun. 1995; 209: 1082-1088Crossref PubMed Scopus (42) Google Scholar, 15Yang Y. Farese R.V. FEBS Lett. 1993; 333: 287-290Crossref PubMed Scopus (23) Google Scholar). Blank values were obtained by substituting a nonimmune antibody preparation instead of the anti-ERK2 antibodies, or by omitting myelin basic protein substrate (results were similar). Except for greater relative effects of insulin, results obtained with this ERK immune complex assay were similar in most aspects to those obtained in assays of total mitogen-activated protein kinase activity observed in crude cell extracts (5Standaert M.L. Bandyopadhyay G. Farese R.V. Biochem. Biophy. Res. Commun. 1995; 209: 1082-1088Crossref PubMed Scopus (42) Google Scholar, 15Yang Y. Farese R.V. FEBS Lett. 1993; 333: 287-290Crossref PubMed Scopus (23) Google Scholar). Differences in absolute 32P-incorporation values between individual experiments reflect variations in amounts of cell extracts immunoprecipitated and specific activity of [γ-32P]ATP used, but relative effects of insulin and other agonists were comparable. In most cases, the actual data from individual experiments are depicted, but in all cases similar findings were observed in repeat experiments. As depicted in representative blots in Fig. 1, and as quantified in multiple samples in TableI, treatments with insulin and PI3K and PKC-ζ inhibitors, wortmannin and the myristoylated PKC-ζ pseudosubstrate, did not have significant effects on the levels of ERK1 and ERK2, or their ratios, in these ERK2 immunoprecipitates, as determined by blotting with a rabbit polyclonal antiserum that recognizes both ERK1 and ERK2 in Western analyses. It may therefore be surmised that these ERK2 assays actually reflected activities of both ERK1 and ERK2, and our present finding of insulin effects on both ERK1 and ERK2 in these immunoprecipitates is in keeping with our previous findings, which showed that insulin activates both p44 ERK1 and p42 ERK2 in rat adipocytes, as determined following their electrophoretic resolution and assay in myelin basic protein-containing gels (15Yang Y. Farese R.V. FEBS Lett. 1993; 333: 287-290Crossref PubMed Scopus (23) Google Scholar).Table ILevels of immunoprecipitable ERK1 and ERK2 following treatment of rat adipocytes with insulin, wortmannin, and/or myristoylated PKC-ζ pseudosubstrateTreatmentERK1 relative valuesERK1 insulin/controlERK2 relative valuesERK2 insulin/controlERK2/ERK1Control1.00 ± 0.241.00 ± 0.211.69 ± 0.40Insulin0.89 ± 0.160.89 ± 0.161.07 ± 0.281.07 ± 281.62 ± 0.24Wortmannin0.87 ± 0.201.07 ± 0.291.77 ± 0.31Wortmannin + insulin1.13 ± 0.271.32 ± 171.34 ± 0.341.35 ± 181.84 ± 0.25MYR-PKC-ζ-PS1.14 ± 0.431.40 ± 0.501.72 ± 0.23MYR-PKC-ζ-PS + insulin1.09 ± 0.490.89 ± 101.15 ± 0.520.79 ± 101.42 ± 0.22Adipocytes were treated first without inhibitors or with 100 nm wortmannin for 15 min, or with 50 μM myristoylated (MYR) PKC-ζ pseudosubstrate (PS) for 60 min, and second with or without 10 nm insulin for 10 min. ERK was immunoprecipitated with anti-ERK2 mouse monoclonal antibody, and precipitates were resolved by SDS-polyacrylamide gel electrophoresis and blotted with a rabbit polyclonal anti-ERK antiserum that recognizes both ERK1 and ERK2. After chemiluminescence development, p44 ERK1 and p42 ERK2 bands were quantitated with Bio-Rad molecular analyst chemiluminescence/32P imaging system. Values are mean ± S.E. of four determinations. Note that the mean control value was set at a relative value of 1.00, and four blots were compared simultaneously on the same Bio-Rad molecular analyst chemiluminescence imaging screen, thus allowing the direct comparison of 4 sets of immunoprecipitations, with each set containing each treatment. See Fig.1 for representative blots. Open table in a new tab Adipocytes were treated first without inhibitors or with 100 nm wortmannin for 15 min, or with 50 μM myristoylated (MYR) PKC-ζ pseudosubstrate (PS) for 60 min, and second with or without 10 nm insulin for 10 min. ERK was immunoprecipitated with anti-ERK2 mouse monoclonal antibody, and precipitates were resolved by SDS-polyacrylamide gel electrophoresis and blotted with a rabbit polyclonal anti-ERK antiserum that recognizes both ERK1 and ERK2. After chemiluminescence development, p44 ERK1 and p42 ERK2 bands were quantitated with Bio-Rad molecular analyst chemiluminescence/32P imaging system. Values are mean ± S.E. of four determinations. Note that the mean control value was set at a relative value of 1.00, and four blots were compared simultaneously on the same Bio-Rad molecular analyst chemiluminescence imaging screen, thus allowing the direct comparison of 4 sets of immunoprecipitations, with each set containing each treatment. See Fig.1 for representative blots. Rat adipocytes were transiently transfected using an electroporation method described previously (14Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar,17Bandyopadhyay G. Standaert M.L. Kikkawa U. Ono Y. Moscat J. Farese R.V. Biochem. J. 1999; 337: 461-470Crossref PubMed Scopus (129) Google Scholar). In brief, 0.4 ml of adipocyte was suspended in an equal volume of sterile Dulbecco's modified Eagle's medium containing 5% bovine serum albumin and 3.3 μg of pCMV5 encoding MYC-tagged ERK2 or 1 μg of pCEP4 encoding hemagglutinin (HA)-tagged ERK2 (both kindly supplied by Dr. Melanie Cobb), along with, as indicated in individual experiments: (a) 6.7 μg of pCDNA3 encoding dominant-negative Δp85 PI3K subunit mutant (kindly supplied by Dr. Masato Kasuga; see Ref. 18Sakaue H. Haras K. Noguchi T. Matozaki T. Kotani K. Ogawa W. Yonezawa K. Waterfield M.D. Kasuga M. J. Biol. Chem. 1995; 270: 11304-11309Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar); (b) 9 μg of pRSV encoding N17 dominant-negative or V12 constitutive RAS mutants (both kindly supplied by Dr. Jane Reusch); (c) 6.7 μg of pCEP4 encoding dominant-negative mutant forms of c-RAF-1 (a truncated form containing the N-terminal RAS-binding domain of c-RAF-1 that consequently inhibits RAS-dependent activation of endogenous ERK2) or MEKK1 (kinase-inactive, D1369A mutant) (both kindly supplied by Dr. Melanie Cobb); (d) 9 μg of pCDNA3 encoding wild-type (WT), constitutive, or kinase-inactive (KI) PKC-ζ (see Refs. 14Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar and 17Bandyopadhyay G. Standaert M.L. Kikkawa U. Ono Y. Moscat J. Farese R.V. Biochem. J. 1999; 337: 461-470Crossref PubMed Scopus (129) Google Scholar); (e) 9 μg of pCMVS encoding mutant T410A PKC-ζ (kindly supplied by Dr. Alex Toker, see Ref. 19Chou M.M. Hou W. Johnson J. Grahams L.K. Lee M.H. Chen C.S. Newton A.C. Schaffhause B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Abstract Full Text Full Text PDF PubMed Google Scholar); (f) 9 μg of pCDNA3 encoding WT or KI (K110N mutant) PDK-1 (both kindly supplied by Dr. Alex Toker; see Ref.19); (g) 6.7 μg of pSRα encoding WT or a dominant-negative SOS that interferes with GTP/GDP exchange in RAS (kindly supplied by Dr. Masato Kasuga, see Ref. 20Takeda H. Matozaki T. Takada T. Noguchi T. Yamao T. Tsuda M. Ochi F. Fukunaga K. Inagaki K. Kasuga M. EMBO J. 1999; 18: 386-395Crossref PubMed Scopus (137) Google Scholar); or (h) the vector alone. The amount of plasmid DNA used for transfection was kept constant in all samples by varying the amount of insert-free vector. After electroporation, cells were incubated overnight to allow time for expression, and then washed and suspended in glucose-free KRP medium and incubated for 10 min with or without 10 nm insulin. After incubation, cells were sonicated and MYC- or HA-tagged ERK2 was immunoprecipitated with rabbit polyclonal anti-MYC antiserum (Upstate Biotechnologies Inc., Lake Placid, NY) or mouse monoclonal anti-HA antibodies (Covance, Richmond, CA), respectively, and assayed for ERK-dependent myelin basic protein phosphorylation, as described above. As shown in the immunoblots depicted in Fig. 1, and, as may be surmised from observing comparable levels of basal enzyme activity of epitope-tagged ERK2 in various co-transfection groups (see Figs. Figure 3, Figure 4, Figure 5), the transfection of dominant-negative forms of SOS, RAS, RAF, MEKK1, Δp85 PI3K, and various forms of PKC-ζ and PDK-1 had relatively little or no significant effect on the levels of immunoprecipitable epitope-tagged ERK2. Also note that only ERK2 was recovered in immunoprecipitates obtained with anti-HA and anti-MYC antibodies (Fig.1). Initially, we used inhibitors to identify factors required for insulin-induced activation of immunoprecipitable ERK2 in rat adipocytes. As seen in Fig. 2, PI3K inhibitors, wortmannin (100 nm) and LY294002 (100 μm) (i.e. in concentrations required to largely inhibit insulin-stimulated glucose transport in the rat adipocyte), and the MEK1 inhibitor, PD098059 (10 μm, which did not inhibit insulin-stimulated glucose transport), inhibited insulin-stimulated increases in immunoprecipitable ERK. Of particular interest, the cell-permeable myristoylated PKC-ζ pseudosubstrate inhibited insulin-induced increases in immunoprecipitable ERK activity over a concentration range comparable with that which is effective in inhibiting PKC-ζ (14Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). In this regard, diacylglycerol-dependent PKCs are not required for insulin-induced activation of ERK in rat adipocytes (see Ref. 15Yang Y. Farese R.V. FEBS Lett. 1993; 333: 287-290Crossref PubMed Scopus (23) Google Scholar; presently, we also confirmed that phorbol ester-induced PKC down-regulation inhibited the acute effects of phorbol esters but did not inhibit insulin-induced activation of immunoprecipitable ERK; data not shown), and it is therefore clear that the PKC-ζ pseudosubstrate did not exert its effects through inhibition of diacylglycerol-dependent PKCs. These findings with inhibitors suggested that PI3K and PKC-ζ (or PKC-λ, which is 72% homologous to PKC-ζ and has an identical pseudosubstrate sequence), as well as MEK1, are required for insulin-induced activation of ERK in rat adipocytes. Further evidence implicating PI3K in insulin-induced activation of ERK was obtained in experiments in which rat adipocytes were transiently co-transfected with plasmids encoding MYC-tagged ERK2 and a dominant-negative mutant form of the p85 subunit of PI3K, Δp85 (18Sakaue H. Haras K. Noguchi T. Matozaki T. Kotani K. Ogawa W. Yonezawa K. Waterfield M.D. Kasuga M. J. Biol. Chem. 1995; 270: 11304-11309Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). As seen in Fig. 3, insulin-induced activation of MYC-ERK2 was inhibited by dominant-negative Δp85, a mutant form of the p85 subunit of PI3K that interacts with phosphotyrosine (pY) residues on activated forms of IRS family members but is unable to transmit activating signals to the p110 catalytic subunit of PI3K (18Sakaue H. Haras K. Noguchi T. Matozaki T. Kotani K. Ogawa W. Yonezawa K. Waterfield M.D. Kasuga M. J. Biol. Chem. 1995; 270: 11304-11309Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These findings therefore suggested that the p85 regulatory subunit, as well as the p110 catalytic subunit (which is directly inhibited by wortmannin and LY294002), of PI3K was required for ERK2 activation, presumably reflecting a need for activation of the SH2 domain of the p85 subunit by pYXXM-containing proteins such as IRS (this assumes that Δp85 neither binds nor inhibits the p110 subunit of PI3K). In addition to PI3K, we found that SOS, RAS, and RAF were required for insulin-induced activation of ERK2 in rat adipocytes. As seen in Fig.3, transient transfection of dominant-negative mutant forms of SOS, RAS, and c-RAF-1 inhibited the activation of co-transfected HA- or MYC-tagged ERK2 by insulin; in addition, constitutively active RAS markedly stimulated HA-ERK2 activity. In contrast to the c-RAF-1 mutant, transfection of a dominant-negative kinase-inactive mutant form of MEKK1, which like RAF and PI3K (21Giglione C. Parmeggiani A. J. Biol. Chem. 1998; 273: 34737-34744Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) can interact with RAS (22Russell M. Lange-Carter C.A. Johnson G.L. J. Biol. Chem. 1995; 270: 11757-11760Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), had no effect on basal or insulin-stimulated MYC-ERK2 (Fig. 3). Also, in contrast to inhibitory effects of dominant-negative RAS on insulin-induced activation of HA-ERK2, this RAS mutant did not inhibit the acute activating effects of phorbol esters on HA-ERK2 (data not shown), which may in some cell types occur independently of RAS (23Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1662) Google Scholar). Thus, the inhibitory effects of both dominant-negative RAS and c-RAF-1 on insulin-induced activation of HA-ERK2 appeared to be specific. The above findings suggested that PI3K along with SOS, RAS, c-RAF-1, and MEK1 was required for insulin-induced activation of ERK2 in the rat adipocyte. Because PKC-ζ (and/or λ) is known to serve as an effector of PI3K during insulin action in rat adipocytes (14Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar) and other cells (24Bandyopadhyay G. Standaert M.L. Zhao L., Yu, B. Avignon A. Galloway L. Karnama P. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 2551-2558Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 25Mendez R. Kollmorgen G. White M.F. Rhoads R.E. Cell Biol. 1997; 17: 5184-5192Google Scholar, 26Bandyopadhyay G. Standaert M.L. Galloway L. Moscat J. Farese R.V. Endocrinology. 1997; 138: 4721-4731Crossref PubMed Scopus (209) Google Scholar, 27Kotani K. Ogawa W. Matsumoto M. Kitamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar), and in view of the above-described inhibitor studies, we tested the possibility that PKC-ζ may function downstream of PI3K during ERK activation by transiently co-transfecting rat adipocytes with plasmids encoding HA-ERK2 and various forms of PKC-ζ. As seen in Fig. 4, whereas WT PKC-ζ had no effect on HA-ERK2 activity, both a KI form of PKC-ζ (K271N mutant) and an activation-resistant form of PKC-ζ (T410A mutant that cannot be activated by PDK-1; see Refs. 19Chou M.M. Hou W. Johnson J. Grahams L.K. Lee M.H. Chen C.S. Newton A.C. Schaffhause B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Abstract Full Text Full Text PDF PubMed Google Scholar and 28LeGood J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (972) Google Scholar) markedly inhibited insulin-stimulated HA-ERK2 activity but had little effect on basal or phorbol ester-stimulated HA-ERK2 activity. Moreover, the inhibitory effect of KI-PKC-ζ could be reversed (or prevented) by co-transfecting plasmid encoding WT-PKC-ζ, which alone had no effect on basal or insulin-stimulated ERK2. These findings suggested that the point-mutation per se in KI-PKC-ζ was responsible for its inhibitory effects on insulin-induced activation of ERK2 and further implied that the kinase activity of PKC-ζ is specifically required, presumably to phosphorylate a presently undefined substrate that is required for subsequent ERK2 activation in rat adipocytes. Whereas mutant forms of PKC-ζ inhibited insulin-induced activation of HA-ERK2, constitutive PKC-ζ provoked insulin-like increases in HA-ERK2 activity, even in the absence of insulin (Fig. 4). Further stimulatory effects of insulin on ERK2 activity in cells expressing constitutive PKC-ζ (Fig. 4) were also observed, and this may reflect the activation of endogenous PKC-ζ, or the fact that insulin can provoke further increases in activity of "constitutive" PKC-ζ. 2M. P. Sajan, M. L. Standaert, G. Bandyopadhyay, M. J. Quon, T. R. Burke, Jr., and R. V. Farese, unpublished observations. Because PDK-1, in conjunction with PI3K-dependent increases in PI-3,4,5-(PO4)3, has been reported to transmit activating signals from PI3K to PKC-ζ (19Chou M.M. Hou W. Johnson J. Grahams L.K. Lee M.H. Chen C.S. Newton A.C. Schaffhause B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Abstract Full Text Full Text PDF PubMed Google Scholar, 28LeGood J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (972) Google Scholar) (note that we have confirmed that PDK-1 and its target in PKC-ζ, threonine-410, are required for PKC-ζ activation by insulin in rat adipocytes; see Ref.29Bandyopadhyay G. Standaert M.L. Sajan M.P. Karnitz L.M. Cong L. Quon M.J. Farese R.V. Mol. Endocrinol. 1999; PubMed Google Scholar), we examined the role of PDK-1 in insulin-induced activation of ERK2 in transiently transfected rat adipocytes. As seen in Fig.5, WT-PDK-1 enhanced basal HA-ERK2 activity, and KI-PDK-1 inhibited insulin-stimulated HA-ERK2 activity. Further, the inhibitory effect of KI-PDK-1 on insulin-stimulated ERK2 activation was reversed by co-transfection of WT-PDK-1 (Fig. 5), indicating that its kinase activity (presumably to phosphorylate Thr-410 in PKC-ζ), like that of PKC-ζ, is specifically required for insulin-induced activation of ERK2. Our findings suggested that PI3K, PDK-1, and PKC-ζ, along with SOS, RAS, c-RAF-1, and MEK1 were required for ERK2 activation during insulin stimulation of rat adipocytes. In this regard, RAS is known to bind to the p110 subunit of PI3K (10Kodaki T. Woscholski R. Hallberg B. Rodriquez-Viciana P. Downward J. Parker P.J. Curr. Biol. 1994; 4: 798-806Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 11Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1726) Google Scholar), and we presently found that both the p110 and p85 subunits of PI3K were recovered in RAS immunoprecipitates prepared from lysates of rat adipocytes (Fig.6). However, we did not observe any effects of insulin on (a) p85 or p110 PI3K subunit levels in RAS immunoprecipitates or (b) PI3K enzyme activity recovered in RAS immunoprecipitates (Fig. 6). Moreover, as discussed above, the inhibitory effects of dominant-negative Δp85 on insulin-induced activation of ERK2 suggested that the activation of PI3K that is relevant to ERK activation requires input from a factor that is capable of activating the p85 subunit of PI3K, e.g. an IRS family member. Collectively, these findings suggested that RAS may serve in the localization but not in the activation of PI3K. Finally, we found that inhibitors of tyrosine kinase and the GRB2 SH2 domain, viz., genistein and a Nα-oxalyl-tripeptide-pY mimetic (see above and Ref.16), respectively, also inhibited insulin-induced activation of immunoprecipitable ERK in the rat adipocyte (Fig. 7). These findings therefore suggested that a tyrosine kinase-dependent substrate, i.e. IRS and/or SHC, along with GRB2 and SOS, operate upstream of RAS in insulin-induced activation of ERK2 in rat adipocytes. To summarize, our findings suggested that factors in two signaling pathways that are frequently considered to be functionally separate during insulin action, viz., the GRB2/SOS/RAS/RAF pathway and the PI3K/PDK-1/PKC-ζ pathway, are jointly required for insulin-induced activation of ERK in rat adipocytes. Although there are still gaps in our understanding of how these pathways are activated and interact with each other, it seems likely that one or more activated forms of IRS family members acts upon a specific pool of PI3K that operates in conjunction with GRB2/SOS and RAS. This PI3K apparently activates a subset of PDK-1 and PKC-ζ, which in turn may contribute, along with RAS, to the activation of c-RAF-1. This postulation is in keeping with other findings indicating that RAS-dependent activation of RAF apparently requires the phosphorylation of RAF by serine/threonine kinases and subsequent recruitment of a 14·3·3 protein (30Vojtek A.B. Der C.J. J. Biol. Chem. 1998; 273: 19925-19928Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 31Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar, 32Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (387) Google Scholar). Alternatively, despite our negative finding, RAS may activate PI3K (see Refs. 10Kodaki T. Woscholski R. Hallberg B. Rodriquez-Viciana P. Downward J. Parker P.J. Curr. Biol. 1994; 4: 798-806Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar and 11Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1726) Google Scholar), or IRS-stimulated PI3K may activate RAS either directly (12Hu Q. Klippe l A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (517) Google Scholar) or indirectly via GRB2/SOS, as suggested to occur during Gβγ signaling through PI3K and RAS (33Lopez-Ilasaca M. Crespo P. Giuseppe Pellici P. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (628) Google Scholar); however, in the latter case, PI3K is postulated to activate a nonreceptor tyrosine kinase before activating GRB2/SOS (33Lopez-Ilasaca M. Crespo P. Giuseppe Pellici P. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (628) Google Scholar), and in the case of insulin, this would imply an element of redundancy, as it would mean that tyrosine kinase activation is required both before and after PI3K activation. With any of these alternatives, the ability of RAS to bind both PI3K (10Kodaki T. Woscholski R. Hallberg B. Rodriquez-Viciana P. Downward J. Parker P.J. Curr. Biol. 1994; 4: 798-806Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 11Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1726) Google Scholar) and RAF (21Giglione C. Parmeggiani A. J. Biol. Chem. 1998; 273: 34737-34744Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), coupled with localizing and activating effects of PI-3,4,5-(PO4)3, may facilitate the assembly of a functional complex that contains or subsequently recruits RAS, PI3K, PDK-1, PKC-ζ and RAF. Further studies are needed to more precisely define (a) how PI3K and RAS operate with respect to each other and (b) the role of PKC-ζ in insulin-induced activation of ERK. We thank Sara M. Busquets for invaluable secretarial assistance.