Tyrosine phosphorylation of p62(Dok) induced by cell adhesion and insulin: possible role in cell migration

Dok, a 62-kDa Ras GTPase-activating protein (rasGAP)-associated phosphotyrosyl protein, is thought to act as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. Cell adhesion to extracellular matrix proteins induced marked tyrosine phosphorylation of Dok. This adhesion-dependent phosphorylation of Dok was mediated, at least in part, by Src family tyrosine kinases. The maximal insulin-induced tyrosine phosphorylation of Dok required a Src family kinase. A mutant Dok (DokDeltaPH) that lacked its pleckstrin homology domain failed to undergo tyrosine phosphorylation in response to cell adhesion or insulin. Furthermore, unlike the wild-type protein, DokDeltaPH did not localize to subcellular membrane components. Insulin promoted the association of tyrosine-phosphorylated Dok with the adapter protein NCK and rasGAP. In contrast, a mutant Dok (DokY361F), in which Tyr361 was replaced by phenylalanine, failed to bind NCK but partially retained the ability to bind rasGAP in response to insulin. Overexpression of wild-type Dok, but not that of DokDeltaPH or DokY361F, enhanced the cell migratory response to insulin without affecting insulin activation of mitogen-activated protein kinase. These results identify Dok as a signal transducer that potentially links, through its interaction with NCK or rasGAP, cell adhesion and insulin receptors to the machinery that controls cell motility.     

Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.     

Role of Gab1 in heart, placenta, and skin development and growth factor- and cytokine-induced extracellular signal-regulated kinase mitogen-activated protein kinase activation

Gab1 is a member of the Gab/DOS (Daughter of Sevenless) family of adapter molecules, which contain a pleckstrin homology (PH) domain and potential binding sites for SH2 and SH3 domains. Gab1 is tyrosine phosphorylated upon stimulation of various cytokines, growth factors, and antigen receptors in cell lines and interacts with signaling molecules, such as SHP-2 and phosphatidylinositol 3-kinase, although its biological roles have not yet been established. To reveal the functions of Gab1 in vivo, we generated mice lacking Gab1 by gene targeting. Gab1-deficient embryos died in utero and displayed developmental defects in the heart, placenta, and skin, which were similar to phenotypes observed in mice lacking signals of the hepatocyte growth factor/scatter factor, platelet-derived growth factor, and epidermal growth factor pathways. Consistent with these observations, extracellular signal-regulated kinase mitogen-activated protein (ERK MAP) kinases were activated at much lower levels in cells from Gab1-deficient embryos in response to these growth factors or to stimulation of the cytokine receptor gp130. These results indicate that Gab1 is a common player in a broad range of growth factor and cytokine signaling pathways linking ERK MAP kinase activation.     

Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice.

We and others recently generated mice with a targeted disruption of the insulin receptor substrate 1 (IRS-1) gene and demonstrated that they exhibited growth retardation and had resistance to the glucose-lowering effect of insulin. Insulin initiates its biological effects by activating at least two major signalling pathways, one involving phosphatidylinositol 3-kinase (PI3-kinase) and the other involving a ras/mitogen-activated protein kinase (MAP kinase) cascade. In this study, we investigated the roles of IRS-1 and IRS-2 in the biological action in the physiological target organs of insulin by comparing the effects of insulin in wild-type and IRS-1-deficient mice. In muscles from IRS-1-deficient mice, the responses to insulin-induced PI3-kinase activation, glucose transport, p70 S6 kinase and MAP kinase activation, mRNA translation, and protein synthesis were significantly impaired compared with those in wild-type mice. Insulin-induced protein synthesis was both wortmannin sensitive and insensitive in wild-type and IRS-1 deficient mice. However, in another target organ, the liver, the responses to insulin-induced PI3-kinase and MAP kinase activation were not significantly reduced. The amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) was roughly equal to that of IRS-1 (in wild-type mice) in the liver, whereas it only 20 to 30% of that of IRS-1 in the muscles. In conclusion, (i) IRS-1 plays central roles in two major biological actions of insulin in muscles, glucose transport and protein synthesis; (ii) the insulin resistance of IRS-1-deficient mice is mainly due to resistance in the muscles; and (iii) the degree of compensation for IRS-1 deficiency appears to be correlated with the amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) relative to that of IRS-1 (in wild-type mice).     

A functional role for the p62–ERK1 axis in the control of energy homeostasis and adipogenesis

In vivo genetic inactivation of the signalling adapter p62 leads to mature-onset obesity and insulin resistance, which correlate with reduced energy expenditure (EE) and increased adipogenesis, without alterations in feeding or locomotor functions. Enhanced extracellular signal-regulated kinase (ERK) activity in adipose tissue from p62-knockout (p62^−/−) mice, and differentiating fibroblasts, suggested an important role for this kinase in the metabolic alterations of p62^−/− mice. Here, we show that genetic inactivation of ERK1 in p62^−/− mice reverses their increased adiposity and adipogenesis, lower EE and insulin resistance. These results establish genetically that p62 is a crucial regulator of ERK1 in metabolism.     

Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.     

Association of insulin receptor substrate 1 (IRS-1) y895 with Grb-2 mediates the insulin signaling involved in IRS-1-deficient brown adipocyte mitogenesis

We have recently generated immortalized fetal brown adipocyte cell lines from insulin receptor substrate 1 (IRS-1) knockout mice and demonstrated an impairment in insulin-induced lipid synthesis as compared to wild-type cell lines. In this study, we investigated the consequences of IRS-1 deficiency on mitogenesis in response to insulin. The lack of IRS-1 resulted in the inability of insulin-stimulated IRS-1-deficient brown adipocytes to increase DNA synthesis and enter into S/G2/M phases of the cell cycle. These cells showed a severe impairment in activating mitogen-activated protein kinase kinase (MEK1/2) and p42-p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. IRS-1-deficient cells also lacked tyrosine phosphorylation of SHC and showed no SHC-Grb-2 association in response to insulin. The mitogenic response to insulin could be partially restored by enhancing IRS-2 tyrosine phosphorylation and its association with Grb-2 by inhibition of phosphatidylinositol 3-kinase activity through a feedback mechanism. Reconstitution of IRS-1-deficient brown adipocytes with wild-type IRS-1 restored insulin-induced IRS-1 and SHC tyrosine phosphorylation and IRS-1-Grb-2, IRS-1-SHC, and SHC-Grb-2 associations, leading to the activation of MAPK and enhancement of DNA synthesis. Reconstitution of IRS-1-deficient brown adipocytes with the IRS-1 mutant Tyr895Phe, which lacks IRS-1-Grb-2 binding, restored SHC-IRS-1 association and SHC-Grb-2 association. However, the lack of IRS-1-Grb-2 association impaired MAPK activation and DNA synthesis in insulin-stimulated mutant cells. These data provide strong evidence for an essential role of IRS-1 and its direct association with Grb-2 in the insulin signaling pathway leading to MAPK activation and mitogenesis in brown adipocytes.     

Insulin-induced beta-arrestin1 Ser-412 phosphorylation is a mechanism for desensitization of ERK activation by Galphai-coupled receptors

Beta-arrestin1 is an adapter/scaffold for many G protein-coupled receptors during mitogen-activated protein kinase signaling. Phosphorylation of beta-arrestin1 at position Ser-412 is a regulator of beta-arrestin1 function, and in the present study, we showed that insulin led to a time- and dose-dependent increase in beta-arrestin1 Ser-412 phosphorylation, which blocked isoproterenol- and lysophosphatidic acid-induced Ser-412 dephosphorylation and impaired ERK signaling by these G protein-coupled receptor ligands. Insulin treatment also led to accumulation of Ser-412-phosphorylated beta-arrestin1 at the insulin-like growth factor 1 receptor and prevented insulin-like growth factor 1/Src association. Insulin-induced Ser-412 phosphorylation was partially dependent on ERK as treatment with the MEK inhibitor PD98059 inhibited the insulin effect (62% reduction, p = 0.03). Inhibition of phosphatidylinositol 3-kinase by wortmannin did not have a significant effect (9% reduction, p = 0.41). We also found that the protein phosphatase 2A (PP2A) was in a molecular complex with beta-arrestin1 and that the PP2A inhibitor okadaic acid increased Ser-412 phosphorylation. Concomitant addition of insulin and okadaic acid did not produce an additive effect on Ser-412 phosphorylation, suggesting a common mechanism. Small t antigen specifically inhibited PP2A, and in HIRcB cells expressing small t antigen, beta-arrestin1 Ser-412 phosphorylation was increased, and insulin had no further effect. Insulin treatment caused increased beta-arrestin1 Ser-412 phosphorylation, which blocked mitogen-activated protein kinase signaling and internalization by beta-arrestin1-dependent receptors with no effect on beta-adrenergic receptor Gs-mediated cAMP production. These findings provide a new mechanism for insulin-induced desensitization of ERK activation by Galphai-coupled receptors.     

Rap1a deficiency modifies cytokine responses and MAPK-signaling in vitro and impairs the in vivo inflammatory response

Rap1, which is closely related to ras, plays a key role in T-cell receptor (TCR)-signaling. TCR-stimulation without costimulation leads to constitutively activated rap1, which may mediate T-cell anergy via inhibition of ras-dependent induction of extracellular signal-regulated kinases (ERK). This activation is mediated by a second protein kinase b-Raf. Rap1-GTP is thought to activate ERK in a ras-independent manner by binding b-raf. Generally, T cells do not express b-raf while they express the adaptor protein raf-1, which is usually sequestered by rap1 leading to inhibition of ras-mediated ERK activation. In this study, we demonstrate that in rap1-deficient T cells, signaling by the ERK and p38 kinases is increased following activation by different stimuli leading to increased intracellular accumulation and secretion of cytokines. In addition, in a hypersensitivity model rap1-deficient mice demonstrated reduced contact dermatitis compared to wildtype mice, demonstrating the impact of rap1-deficiency on the inflammatory response in vivo.     

PPAR-gamma inhibits ANG II-induced cell growth via SHIP2 and 4E-BP1

The present study evaluated the effects of peroxisome proliferator-activated receptor (PPAR)-gamma activators on ANG II-induced signaling pathways and cell growth. Vascular smooth muscle cells (VSMC) derived from rat mesenteric arteries were treated with ANG II, with/without the AT1 receptor blocker valsartan or the AT2 receptor blocker PD-123319, after pretreatment for 24 h with the PPAR-gamma activators 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) or rosiglitazone. Both 15d-PGJ2 and rosiglitazone decreased ANG II-induced DNA synthesis. Rosiglitazone treatment increased nuclear PPAR-gamma expression and activity in VSMC. However, rosiglitazone did not alter expression of PPAR-alpha/beta, ERK 1/2, Akt, or ANG II receptors. 15d-PGJ2 and rosiglitazone decreased ERK 1/2 and Akt peak activity, both of which were induced by ANG II via the AT1 receptor. Rosiglitazone inhibited ANG II-enhanced phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), as well as Src homology (SH) 2-containing inositol phosphatase 2 (SHIP2). PPAR-gamma activation reduced ANG II-induced growth associated with inhibition of ERK 1/2, Akt, 4E-BP1, and SHIP2. Modulation of these pathways by PPAR-gamma activators may contribute to regression of vascular remodeling in hypertension.     

Grb10 mediates insulin-stimulated degradation of the insulin receptor: a mechanism of negative regulation

Growth factor receptor-bound protein 10 (Grb10) is an adapter protein that interacts with a number of tyrosine-phosphorylated growth factor receptors, including the insulin receptor (IR). To investigate the role of Grb10 in insulin signaling, we generated cell lines in which the expression levels of Grb10 are either overexpressed by stable transfection or suppressed by RNA interference. We found that suppressing endogenous Grb10 expression led to increased IR protein levels, whereas overexpression of Grb10 led to reduced IR protein levels. Altering Grb10 expression levels had no effect on the mRNA levels of IR, suggesting that the modulation occurs at the protein level. Reduced IR levels were also observed in cells with prolonged insulin treatment, and this reduction was inhibited in Grb10-deficient cells. The insulin-induced IR reduction was greatly reversed by MG-132, a proteasomal inhibitor, but not by chloroquine, a lysosomal inhibitor. IR underwent insulin-stimulated ubiquitination in cells, and this ubiquitination was inhibited in the Grb10-suppressed cell line. Together, our results suggest that, in addition to inhibiting IR kinase activity by directly binding to the IR, Grb10 also negatively regulates insulin signaling by mediating insulin-stimulated degradation of the receptor.     

Neuropeptide Y promotes beta-cell replication via extracellular signal-regulated kinase activation

AIMS/HYPOTHESIS: Previous studies have shown that neuropeptide Y (NPY) gene expression and release are increased in hyperphagic ob/ob mice and diabetic rats. Therefore, we hypothesized that orexigenic agent, NPY, has the effect on the obesity and diabetes. To elucidate the relationship, we have studied the regulatory role of NPY on islet cells. METHODS: Isolated islets were incubated with NPY or NPY Y1 receptor specific antagonist, BIBP3226. Proliferation, apoptosis, and Y1 receptor expression were identified by immunohistochemistry. We studied that ERK1/2 mediates the NPY pathway with PD98059 (MAP kinase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), and BIM-1 (protein kinase C inhibitor). After NPY-treated islets were exposed to high glucose, insulin levels were detected. RESULTS: beta-Cell replication was enhanced in a dose-dependent manner, but without any changes on the other cells in islet. NPY Y1 receptors were expressed on islet and NPY induced phosphorylation of ERK1/2 rapidly and transiently. PD98059 (MAPK kinase inhibitor) and BIM-1 (protein kinase C inhibitor) inhibited activation of ERK1/2 by NPY, but wortmannin (phosphatidylinositol 3-kinase inhibitor) did not. Exposure of NPY-treated islets to high glucose showed the decreasing trend of insulin secretion. CONCLUSION/INTERPRETATION: Our data suggest that NPY promotes beta-cell replication via extracellular signal-regulated kinase activation and inhibits glucose-stimulated insulin secretion.     

MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5-BMK1/ERK5 pathway

MEKK2 and MEKK3 are two closely related mitogen-activated protein kinase (MAPK) kinase kinases. The kinase domains of MEKK2 and MEKK3 are nearly identical, although their N-terminal regulatory domains are significantly divergent. By yeast two-hybrid library screening, we have identified MEK5, the MAPK kinase in the big mitogen-activated protein kinase 1 (BMK1)/ERK5 pathway, as a binding partner for MEKK2. MEKK2 expression stimulates BMK1/ERK5 activity, the downstream substrate for MEK5. Compared with MEKK3, MEKK2 activated BMK1/ERK5 to a greater extent, which might correlate with a higher affinity MEKK2-MEK5 interaction. A dominant negative form of MEK5 blocked the activation of BMK1/ERK5 by MEKK2, whereas activation of c-Jun N-terminal kinase (JNK) was unaffected, showing that MEK5 is a specific downstream effector of MEKK2 in the BMK1/ERK5 pathway. Activation of BMK1/ERK5 by epidermal growth factor and H2O2 in Cos7 and HEK293 cells was completely blocked by a kinase-inactive MEKK3 (MEKK3kin(-)), whereas MEKK2kin(-) had no effect. However, in D10 T cells, expression of MEKK2kin(-) but not MEKK3kin(-) inhibited BMK1/ERK5 activity. Two-hybrid screening also identified Lck-associated adapter/Rlk- and Itk-binding protein (Lad/RIBP), a T cell adapter protein, as a binding partner for MEKK2. MEKK2 and Lad/RIBP colocalize at the T cell contact site with antigen-loaded presenting cells, demonstrating cotranslocation of MEKK2 and Lad/RIBP during T cell activation. MEKK3 neither binds Lad/RIBP nor is recruited to the T cell contact with antigen presenting cell. MEKK2 and MEKK3 are differentially associated with signaling from specific upstream receptor systems, whereas both activate the MEK5-BMK1/ERK5 pathway.     

Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life

The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10Delta2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Delta2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10Delta2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health.     

Heparanase Enhances the Insulin Receptor Signaling Pathway to Activate Extracellular Signal-regulated Kinase in Multiple Myeloma

ERK signaling regulates proliferation, survival, drug resistance, and angiogenesis in cancer. Although the mechanisms regulating ERK activation are not fully understood, we previously demonstrated that ERK phosphorylation is elevated by heparanase, an enzyme associated with aggressive behavior of many cancers. In the present study, myeloma cell lines expressing either high or low levels of heparanase were utilized to determine how heparanase stimulates ERK signaling. We discovered that the insulin receptor was abundant on cells expressing either high or low levels of heparanase, but the receptor was highly phosphorylated in heparanase-high cells compared with heparanase-low cells. In addition, protein kinase C activity was elevated in heparanase-high cells, and this enhanced expression of insulin receptor substrate-1 (IRS-1), the principle intracellular substrate for phosphorylation by the insulin receptor. Blocking insulin receptor function with antibody or a small molecule inhibitor or knockdown of IRS-1 expression using shRNA diminished heparanase-mediated ERK activation in the tumor cells. In addition, up-regulation of the insulin signaling pathway by heparanase and the resulting ERK activation were dependent on heparanase retaining its enzyme activity. These results reveal a novel mechanism whereby heparanase enhances activation of the insulin receptor signaling pathway leading to ERK activation and modulation of myeloma behavior.     

Molecular mechanisms of CD200 inhibition of mast cell activation

CD200 and its receptor CD200R are both type I membrane glycoproteins that contain two Ig-like domains. Engagement of CD200R by CD200 inhibits activation of myeloid cells. Unlike the majority of immune inhibitory receptors, CD200R lacks an ITIM in the cytoplasmic domain. The molecular mechanism of CD200R inhibition of myeloid cell activation is unknown. In this study, we examined the CD200R signaling pathways that control degranulation of mouse bone marrow-derived mast cells. We found that upon ligand binding, CD200R is phosphorylated on tyrosine and subsequently binds to adapter proteins Dok1 and Dok2. Upon phosphorylation, Dok1 binds to SHIP and both Dok1 and Dok2 recruit RasGAP, which mediates the inhibition of the Ras/MAPK pathways. Activation of ERK, JNK, and p38 MAPK are all inhibited by CD200R engagement. The reduced activation of these MAPKs is responsible for the observed inhibition of mast cell degranulation and cytokine production. Similar signaling events were also observed upon CD200R engagement in mouse peritoneal cells. These data define a novel inhibitory pathway used by CD200R in modulating mast cell function and help to explain how engagement of this receptor in vivo regulates myeloid cell function.     

Altered Glucose Homeostasis in Mice with Liver-specific Deletion of Src Homology Phosphatase 2

The Src homology 2 domain-containing protein-tyrosine phosphatase Shp2 has been implicated in a variety of growth factor signaling pathways, but its role in insulin signaling has remained unresolved. In vitro studies suggest that Shp2 is both a negative and positive regulator of insulin signaling, although its physiological function in a number of peripheral insulin-responsive tissues remains unknown. To address the metabolic role of Shp2 in the liver, we generated mice with either chronic or acute hepatic Shp2 deletion using tissue-specific Cre-LoxP and adenoviral Cre approaches, respectively. We then analyzed insulin sensitivity, glucose tolerance, and insulin signaling in liver-specific Shp2-deficient and control mice. Mice with chronic Shp2 deletion exhibited improved insulin sensitivity and increased glucose tolerance compared with controls. Acute Shp2 deletion yielded comparable results, indicating that the observed metabolic effects are directly caused by the lack of Shp2 in the liver. These findings correlated with, and were most likely caused by, direct dephosphorylation of insulin receptor substrate (IRS)1/2 in the liver, accompanied by increased PI3K/Akt signaling. In contrast, insulin-induced ERK activation was dramatically attenuated, yet there was no effect on the putative ERK site on IRS1 (Ser⁶¹²) or on S6 kinase 1 activity. These studies show that Shp2 is a negative regulator of hepatic insulin action, and its deletion enhances the activation of PI3K/Akt pathway downstream of the insulin receptor.