Protein Kinase C-ζ and Phosphoinositide-dependent Protein Kinase-1 Are Required for Insulin-induced Activation of ERK 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. 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-32PATP (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-32PATP 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 with each set containing each for representative in a 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 with each set containing each for representative adipocytes were an described 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 G. M.L. Y. Moscat J. Farese R.V. Biochem. J. PubMed Scopus Google Scholar). In of was suspended in an equal of medium containing bovine serum and μg of ERK2 or 1 μg of hemagglutinin ERK2 by along as in individual (a) μg of dominant-negative Δp85 PI3K subunit by Ref. H. K. T. T. K. K. Waterfield M.D. M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google (b) μg of dominant-negative or RAS by μg of dominant-negative of containing the domain of that activation of or by μg of or kinase-inactive PKC-ζ (see 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 and G. M.L. Y. Moscat J. Farese R.V. Biochem. J. PubMed Scopus Google μg of PKC-ζ by Ref. J. A. Curr. Biol. 1998; Full Text Full Text PDF PubMed Google μg of or PDK-1 by μg of or a dominant-negative that with in RAS by Ref. H. T. T. T. T. M. K. K. M. J. PubMed Scopus Google or the The of used for was in all samples by the of After cells were incubated overnight to time for and then washed and suspended in glucose-free medium and incubated for 10 min with or without 10 nm After incubation, cells were sonicated and or ERK2 was immunoprecipitated with rabbit polyclonal antiserum Inc., or mouse monoclonal antibodies CA), and for myelin basic protein as described As shown in the depicted in Fig. 1, and, as may be surmised from observing levels of activity of ERK2 in (see the of dominant-negative of SOS, RAS, RAF, Δp85 PI3K, and of PKC-ζ and PDK-1 or significant on the levels of immunoprecipitable ERK2. that only ERK2 was in immunoprecipitates obtained with and antibodies we used inhibitors to required for insulin-induced activation of immunoprecipitable ERK2 in rat adipocytes. As in Fig. 2, PI3K inhibitors, wortmannin and LY294002 in required to inhibit insulin-stimulated in the rat and the MEK1 inhibitor, PD098059 which did not inhibit insulin-stimulated inhibited insulin-stimulated increases in immunoprecipitable the myristoylated PKC-ζ pseudosubstrate inhibited insulin-induced increases in immunoprecipitable ERK activity a with that which is in PKC-ζ 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, 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 we also confirmed that inhibited the effects of but did not inhibit insulin-induced activation of immunoprecipitable data not and is therefore that the PKC-ζ pseudosubstrate did not effects through of findings with inhibitors suggested that PI3K and PKC-ζ which is to PKC-ζ and has an pseudosubstrate as well as MEK1, are required for insulin-induced activation of ERK in rat adipocytes. PI3K in insulin-induced activation of ERK was obtained in experiments in which rat adipocytes were with ERK2 and a dominant-negative of the subunit of PI3K, Δp85 H. K. T. T. K. K. Waterfield M.D. M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). As in Fig. insulin-induced activation of was inhibited by dominant-negative a of the subunit of PI3K that with phosphotyrosine residues on activated of family members but is to to the subunit of PI3K H. K. T. T. K. K. Waterfield M.D. M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). findings therefore suggested that the as well as the subunit is inhibited by wortmannin and of PI3K was required for ERK2 activation, a for activation of the SH2 domain of the subunit by as (this that Δp85 neither nor the subunit of In addition to PI3K, we found that SOS, RAS, and were required for insulin-induced activation of ERK2 in rat adipocytes. As in transient of dominant-negative of SOS, RAS, and inhibited the activation of or ERK2 by in RAS In to the of a dominant-negative kinase-inactive of which and PI3K C. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) with RAS M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), on or insulin-stimulated in to effects of dominant-negative RAS on insulin-induced activation of this RAS did not inhibit the effects of on not which may in some cell types occur independently of RAS E.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). the effects of both dominant-negative RAS and on insulin-induced activation of to be The findings suggested that PI3K along with SOS, RAS, and MEK1 was required for insulin-induced activation of ERK2 in the rat adipocyte. PKC-ζ is to serve as an of PI3K during insulin action in rat adipocytes 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 G. M.L. B. A. Galloway L. P. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, R. G. Biol. 1997; Scholar, G. M.L. Galloway L. Moscat J. Farese R.V. Endocrinology. 1997; 138: PubMed Scopus Google Scholar, K. M. T. H. Y. K. K. S. M. Mol. Biol. 1998; PubMed Google Scholar), and in of the we the that PKC-ζ may function downstream of PI3K during ERK activation by rat adipocytes with and of PKC-ζ. As in Fig. PKC-ζ on activity, both a of PKC-ζ and an of PKC-ζ that be activated by J. A. Curr. Biol. 1998; Full Text Full Text PDF PubMed Google and Alessi D.R. Cohen P. Parker P.J. Science. 1998; PubMed Scopus Google Scholar) inhibited insulin-stimulated activity but on or the of be by which on or insulin-stimulated ERK2. findings suggested that the in was for effects on insulin-induced activation of ERK2 and that the kinase activity of PKC-ζ is required, to a substrate that is required for ERK2 activation in rat adipocytes. of PKC-ζ inhibited insulin-induced activation of PKC-ζ increases in activity, in the of insulin effects of insulin on ERK2 activity in cells PKC-ζ were also and this may reflect the activation of PKC-ζ, or the fact that insulin increases in activity of PKC-ζ. P. M. L. G. M. J. T. R. Jr., and R. PDK-1, in conjunction with increases in has been reported to from PI3K to PKC-ζ J. A. Curr. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar, Alessi D.R. Cohen P. Parker P.J. Science. 1998; PubMed Scopus Google Scholar) that we have confirmed that PDK-1 and target in PKC-ζ, are required for PKC-ζ activation by insulin in rat G. M.L. L. M.J. Farese R.V. Mol. Endocrinol. PubMed Google Scholar), we the of PDK-1 in insulin-induced activation of ERK2 in rat adipocytes. As in activity, and inhibited insulin-stimulated Further, the of on insulin-stimulated ERK2 activation was by of that kinase activity (presumably to Thr-410 in that of PKC-ζ, is required for insulin-induced activation of ERK2. Our findings suggested that PI3K, PDK-1, and PKC-ζ, along with SOS, RAS, and MEK1 were required for ERK2 activation during insulin of rat adipocytes. In this regard, RAS is to to the 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 found that both the and of PI3K were in RAS immunoprecipitates from of rat adipocytes we did not effects of insulin on (a) or PI3K subunit levels in RAS immunoprecipitates or (b) PI3K activity in RAS immunoprecipitates as the effects of dominant-negative Δp85 on insulin-induced activation of ERK2 suggested that the activation of PI3K that is to ERK activation from a that is of the subunit of PI3K, an family these findings suggested that RAS may serve in the but not in the activation of we found that inhibitors of tyrosine kinase and the GRB2 SH2 viz., genistein and a (see and also inhibited insulin-induced activation of immunoprecipitable ERK in the rat findings therefore suggested that a tyrosine and/or SHC, along with GRB2 and SOS, upstream of RAS in insulin-induced activation of ERK2 in rat adipocytes. our findings suggested that in signaling that are to be during insulin viz., the and the are required for insulin-induced activation of ERK in rat adipocytes. Although are in our of these are activated and with each that or activated of family members a specific of PI3K that in conjunction with and This PI3K activates a of PDK-1 and PKC-ζ, which in may along with RAS, to the activation of This is in keeping with other findings that activation of the phosphorylation of by kinases and of a protein C.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A.J. Full Text Full Text PDF PubMed Scopus Google Scholar, G. Z. J. Nature. 1998; PubMed Scopus Google Scholar). despite our RAS may activate PI3K (see 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 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 PI3K may activate RAS (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 via as suggested to occur during signaling through PI3K and RAS M. P. P. R. Science. 1997; PubMed Scopus Google in the PI3K is to activate a tyrosine kinase M. P. P. R. Science. 1997; PubMed Scopus Google Scholar), and in the of insulin, this an of as mean that tyrosine kinase activation is required both and after PI3K of these the of RAS to 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 C. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), with and effects of may the of a complex that or RAS, PI3K, PDK-1, PKC-ζ and studies are to (a) PI3K and RAS with to each other and (b) the of PKC-ζ in insulin-induced activation of M. for