Insulin stimulates the activity of mitogen-activated protein kinase (MAPK) via its upstream activator, MAPK kinase (MEK), a dual specificity kinase that phosphorylates MAPK on threonine and tyrosine. The potential role of MAPK activation in insulin action was investigated with the specific MEK inhibitor PD98059. Insulin stimulation of MAPK activity in 3T3-L1 adipocytes (2.7-fold) and L6 myotubes (1.4-fold) was completely abolished by pretreatment of cells with the MEK inhibitor, as was the phosphorylation of MAPK and pp90Rsk, and the transcriptional activation of c-fos. Insulin receptor autophosphorylation on tyrosine residues and activation of phosphatidylinositol 3'-kinase were unaffected. Pretreatment of cells with PD98059 had no effect on basal and insulin-stimulated glucose uptake, lipogenesis, and glycogen synthesis. Glycogen synthase activity in extracts from 3T3-L1 adipocytes and L6 myotubes was increased 3-fold and 1.7-fold, respectively, by insulin. Pretreatment with 10 microM PD98059 was without effect. Similarly, the 2-fold activation of protein phosphatase 1 by insulin was insensitive to PD98059. These results indicate that stimulation of the MAPK pathway by insulin is not required for many of the metabolic activities of the hormone in cultured fat and muscle cells.

Insulin stimulation of differentiated 3T3-L1 adipocytes or Chinese hamster ovary cells expressing high levels of the insulin receptor resulted in a time-dependent decrease in the electrophoretic mobility of SOS on sodium dodecyl sulfate-polyacrylamide gels. The reduction in SOS mobility was completely reversed by alkaline phosphatase treatment, and the in vitro phosphorylation of SOS by mitogen-activated protein kinase resulted in a decrease of electrophoretic mobility identical to that following in vivo insulin stimulation. Immunoprecipitation of Grb2 followed by SOS immunoblotting demonstrated a disassociation of the SOS-Grb2 complex that paralleled the decrease in SOS electrophoretic mobility. Similarly, SOS immunoprecipitation followed by Grb2 immunoblotting also indicated an uncoupling of the SOS-Grb2 complex. Further, incubation of wholecell extracts with glutathione-S-transferase-Grb2 fusion proteins demonstrated that insulin stimulation resulted in a decreased affinity of SOS for Grb2. In contrast, the disassociation of SOS from Grb2 did not affect the interactions between Grb2 and tyrosine-phosphorylated Shc. In addition to insulin, several other agents which activate the mitogen-activated protein kinase pathway (platelet-derived growth factor, serum, and phorbol ester) also resulted in the uncoupling of the SOS-Grb2 complex. Consistent with these results, expression of v-ras and v-raf resulted in a constitutive decrease in the association between SOS and Grb2. Together, these data suggest a molecular mechanism accounting for the transient activation of ras due to the uncoupling of the SOS-Grb2 complex following SOS phosphorylation.A common pathway for the activation of mitogen-activated protein (MAP) kinase has recently been established for several tyrosine kinase receptors including the insulin, platelet-derived growth factor (PDGF), and epidermal growth factor receptors (3,23,32,34). In the case of the insulin receptor (IR), activation of the receptor intrinsic tyrosine kinase activity was originally demonstrated to enhance tyrosine phosphorylation of a 185-kDa protein termed IRS1 for IR substrate 1 (22, 47, 51). IRS1 contains multiple tyrosine phosphorylation acceptor sites which, when phosphorylated, create specific recognition motifs for src homology 2 (SH2) domain-containing proteins (55). These include the p85 regulatory subunit of the phosphatidylinositol 3-kinase, the protein tyrosine-specific phosphatase Syp, and the small adaptor proteins Nck and Grb2 (26,46).In addition to IRS1, the SH2 domain-containing ␣2 collagen-related proteins (Shc) have been identified as proximal targets for several growth factor tyrosine kinases, including the IR (38). The Shc family consists of three related proteins, with the 46-and 52-kDa species resulting from alternative usage of two distinct translation initiation sites within the same transcript and the 66-kDa species most likely arising from an alternatively spliced message (36). In contrast to IRS1, the Shc proteins are tyrosine phosphorylated on a single ...

Activation of Ras by the exchange of bound GDP for GTP is predominantly catalyzed by the guanylnucleotide exchange factor SOS. Receptor tyrosine kinases increase Ras-GTP loading by targeting SOS to the plasma membrane location of Ras through the small adaptor protein Grb2. However, despite the continuous stimulation of receptor tyrosine kinase activity, Ras activation is transient and, in the case of insulin, begins returning to the GDP-bound state within 5 min. We report here that the cascade of serine kinases activated directly by Ras results in a mitogen-activated protein kinase kinase (MEK)-dependent phosphorylation of SOS and subsequent disassociation of the Grb2-SOS complex, thereby interrupting the ability of SOS to catalyze nucleotide exchange on Ras. These data demonstrate a molecular feedback mechanism accounting for the desensitization of Ras-GTP loading following insulin stimulation.Previous studies have demonstrated that insulin stimulation of the insulin receptor tyrosine kinase results in Ras activation and subsequent downstream stimulation of the Raf/MEK/ ERK 1 pathway (1-3). The activation of Ras occurs predominantly through the tyrosine phosphorylation of Shc followed by the association with the Grb2-SOS complex (4, 5). However, Ras activation is transient and rapidly returns to the inactive state despite continuous activation of the insulin receptor tyrosine kinase and prolonged Shc tyrosine phosphorylation (6, 7). Since insulin does not affect Ras-GTPase activating protein activity and/or targeting (8, 9), the mechanism responsible for the desensitization of Ras has remained obscure.Recently it has been reported that stimulation of several cell types with growth factors and other mitogenic agents results in the serine/threonine phosphorylation of SOS (10, 11). In addition, SOS phosphorylation precedes an insulin-dependent disassociation of the Grb2-SOS complex (7, 12). The insulin time dependence of the SOS phosphorylation and uncoupling of Grb2 from SOS was consistent with the desensitization phase of Ras inactivation. To determine whether the ERK pathway is involved in this event, we used two independent approaches to inhibit MEK activity and, hence, ERK activation. In this study we demonstrate that prevention of insulin-stimulated SOS phosphorylation and subsequent disassociation of the Grb2-SOS complex results in a prolongation of Ras activation. EXPERIMENTAL PROCEDURESCell Culture-Chinese hamster ovary cells expressing the human insulin receptor (CHO/IR) and 3T3L1 adipocytes were isolated and cultured as described previously (7). Cells were incubated for 16 h in serum-free media and then pretreated for 1 h with vehicle (0.5% dimethyl sulfoxide) or 100 M PD98059. The cells were then incubated with and without 100 nM insulin for various times, followed by lysis in 50 mM Hepes, pH 7.8, 1% Triton X-100, 2.5 mM EDTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium vanadate, 2 M pepstatin, 0.5 trypsin inhibitory unit of aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 10 M le...

The insulin-responsive aminopeptidase (IRAP) is a constituent of the vesicles that contain the insulin-regulated glucose transporter (Glut4). Like Glut4, IRAP translocates to the cell surface in response to insulin. Microinjection into 3T3-L1 adipocytes of a glutathione S-transferase (GST) fusion protein containing the cytosolic portion of IRAP (GST-IRAP-(1-109)), resulted in translocation of Glut4 to the cell surface. Immunostaining of 3T3-L1 adipocytes for Glut4 showed that the percentage of cells with substantial cell surface Glut4 was 10% in unstimulated cells, 8% following injection of GST, and 27% following injection of GST-IRAP-(1-109). Increased cell surface Glut4 occurred within 5-10 min following injection and was maintained for at least 4 h. A fusion protein containing only 28 amino acids from IRAP (GST-IRAP-(55-82)) was as effective in increasing cell surface Glut4 as stimulation with 100 nM insulin (44% versus 43%, respectively). In contrast to insulinstimulated Glut4 translocation, the redistribution of Glut4 following injection of GST-IRAP-(55-82) was not blocked by wortmannin or co-injection with a SH2 domain from the regulatory subunit of phosphatidylinositol 3-kinase. These data suggest that the amino terminus of IRAP interacts with a retention/sorting protein that also regulates the distribution of Glut4 in insulin-responsive cells.

Insulin activation of Ras is mediated by the plasma membrane targeting of the guanylnucleotide exchange factor SOS associated with the small adapter protein Grb2. SOS also lies in an insulin-stimulated feedback pathway in which the serine/threonine phosphorylation of SOS results in disassociation of the Grb2-SOS complex thereby limiting the extent of Ras activation. To examine the relative role of the mitogen-activated protein kinases in the feedback phosphorylation of SOS we determined the signaling specificity of insulin, osmotic shock, and anisomycin to activate the ERK (extracellular-signal regulated kinase) and JNK (c-Jun kinase) pathways. In Chinese hamster ovary cells expressing the human insulin receptor and murine 3T3L1 adipocytes, insulin specifically activated ERK with no significant effect on JNK, whereas anisomycin specifically activated JNK but was unable to activate ERK. In contrast, osmotic shock was equally effective in the activation of both kinase pathways. Insulin and osmotic shock, but not anisomycin, resulted in SOS phosphorylation and disassociation of the Grb2-SOS complex, demonstrating that the JNK pathway was not involved in the insulin-stimulated feedback uncoupling of the Grb2-SOS complex. Both the insulin and osmotic shock-induced activation of ERK was prevented by treatment of cells with the specific MEK inhibitor (PD98059). However, expression of dominant-interfering Ras (N17Ras) inhibited the insulin-but not osmotic shock-stimulated phosphorylation of ERK and SOS. These data demonstrate that activation of the ERK pathway, but not JNK, is responsible for the feedback phosphorylation and disassociation of the Grb2-SOS complex.The mitogen-activated or extracellular-signal regulated kinases (ERK1 1 and ERK2) are proline-directed serine/threonine kinases that phosphorylate a number of cytosolic and nuclear transcription factors (1). Recently, one complete pathway linking receptor tyrosine kinases to the activation of ERK has been established (2, 3). In this pathway, receptor tyrosine kinase activation results in the tyrosine phosphorylation of the receptor itself as well as the proximal cytosolic substrate Shc (4). Receptor autophosphorylation and/or Shc phosphorylation generates docking sites for the src homology 2 (SH2) domain of the 25-kDa adapter protein Grb2 (5). Grb2 also contains two SH3 domains which are responsible for association with the Ras guanylnucleotide exchange factor SOS (6, 7). Thus, the tyrosine phosphorylation of transmembrane receptors and/or Shc results in the formation of a ternary complex (i.e. Shc-Grb2-SOS) that targets SOS to the plasma membrane location of Ras (8, 9). In this manner, SOS can effect the exchange of GDP for GTP on Ras. Once in the activated GTP-bound state, Ras associates with members of the Raf family of serine/threonine kinases (10 -12). Activated Raf functions as an upstream kinase for the dual specificity kinase MEK which phosphorylates and stimulates ERK activity providing an important bifurcation point for the regulation of metabolic, tr...