Sam68 is a docking protein linking GAP and PI3K in insulin receptor signaling

The 68 kDa Src substrate associated during mitosis (Sam68) is an RNA binding protein with Src homology (SH) 2 and 3 domain binding sites. We have recently found that Sam68 is a substrate of the insulin receptor (IR) and that Tyr-phosphorylated Sam68 associates with the SH2 domains of p85 PI3K. In the present work, using HTC-IR cells, we have found that insulin stimulation promotes the relocalization of Sam68 from the nucleus to the cytoplasm, and we have further studied the role of Sam68 in insulin receptor signaling complexes, by co-precipitating experiments. Thus, Sam68 is co-precipitated with p85 PI3K, IRS-1 and IR. The association of Sam68 with these complexes is mediated by the SH2 domains of PI3K. Moreover, we have found that Sam68 is a p120GAP associated protein after Tyr-phosphorylation by the IR. This association is mediated by the SH2 domains of GAP (preferentially the C-terminal SH2). Thus, Sam68 is linking p120GAP to PI3K signaling pathway. In fact, PI3K activity was increased in both anti-Sam68 and anti-GAP immmunoprecipitates upon insulin stimulation. We propose that the recruitment of the docking protein Sam68 to the PI3K pathway may serve to allow the association of other signaling molecules, i.e. p120GAP. In this way, these signaling complexes may modulate other signaling cascades of IR, such as p21Ras pathway.     

PI 3-kinase activation in BCR/abl-transformed hematopoietic cells does not require interaction of p85 SH2 domains with p210 BCR/abl

BCR/abl is a chimeric oncogene implicated in the pathogenesis of human chronic myelogenous leukemia. Expression of the BCR/abl gene induces hematologic malignancies in transgenic mice and transformation of interleukin-3-dependent hematopoietic cells. The mechanism of BCR/abl- mediated transformation of hematopoietic cells is poorly understood and involves activation of at least two signaling pathways, p21ras and PI 3- kinase. Here we report that PI 3,4-P2 and PI 3,4,5-P3, the enzymatic products of PI 3-kinase, accumulate in metabolically labeled transformed hematopoietic cells, in contrast to our previous report on the lack of accumulation of PI 3-kinase products in nontransformed NIH 3T3 fibroblasts that express p210 BCR/abl. Transformed cells also have increased PI 3-kinase activity in total cell extracts and membrane fractions. Activation of PI 3-kinase occurs by occupancy of SH2 domains of PI 3-kinase regulatory subunit, p85, by phosphorylated YXXM motifs. Therefore, we investigated whether BCR/abl binds to p85 and whether this binding is mediated by interaction of p85 SH2 domains with YXXM motif of BCR/abl. Association of p210 BCR/abl with p85 in immune complexes and with p85 SH2 domains was evident in hematopoietic cells that express the wt p210 BCR/abl. However, the binding of BCR/abl to p85 SH2 domains was abolished in cells expressing mutant, temperature- sensitive (ts) p210 BCR/abl in which the tyrosine in the YXXM motif of p210 BCR/abl was replaced by histidine. Despite lack of direct interaction with p85 SH2 domains, expression of ts p210 BCR/abl resulted in rapid, time-dependent activation of total and membrane- associated PI 3-kinase and increased PI 3-kinase activity in anti-P-tyr and anti-abl immunoprecipitates. These data suggest that BCR/abl- induced activation of PI 3-kinase in hematopoietic cells does not require binding of p85 SH2 domains to BCR/abl gene product and involves interaction with other tyrosine phosphorylated intermediate proteins.     

Rescuing 3T3-L1 adipocytes from insulin resistance induced by stimulation of Akt-mammalian target of rapamycin/p70 S6 kinase (S6K1) pathway and serine phosphorylation of insulin receptor substrate-1: effect of reduced expression of p85alpha subunit of phosphatidylinositol 3-kinase and S6K1 kinase

Phosphorylation of insulin receptor substrate-1 (IRS-1) on serine residues has been recognized as a mechanism responsible for a diminution of insulin action and insulin resistance. Potential approaches to improve insulin sensitivity may include interference with and/or reduction in expression of certain signaling intermediates that participate in the pathogenesis of insulin resistance. In this study, we transduced fully differentiated 3T3-L1 adipocytes with a constitutively active myristoylated Akt that led to hyperactivation of mammalian target of rapamycin and p70 S6 kinase (S6K1), increased serine phosphorylation of IRS-1, and reduction in insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and glucose transport. We then reduced expression of the PI 3-kinase regulatory subunit, p85alpha, or expression of S6K1 kinase using small interfering RNA transfections, which led to a reduction in p85alpha expression of 70% at 48 h (P < 0.05) and S6K1 of 49% (P < 0.05). Reduction in expression of either p85alpha or S6K1 achieved with small interfering RNA in the presence of myristoylated Akt rescued 3T3-L1 adipocytes from the insulin resistance induced by serine phosphorylation of IRS-1 and completely restored insulin-stimulated activation of PI 3-kinase and glucose uptake. We conclude that reduction in expression of p85alpha or S6K1 could represent therapeutic targets to mitigate insulin resistance.     

IRS-1 activates phosphatidylinositol 3''-kinase by associating with src homology 2 domains of p85.

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains.     

Activation of eNOS in rat portal hypertensive gastric mucosa is mediated by TNF-alpha via the PI 3-kinase-Akt signaling pathway

Activation of endothelial nitric oxide synthase (eNOS) in portal hypertensive (PHT) gastric mucosa leads to hyperdynamic circulation and increased susceptibility to injury. However, the signaling mechanisms for eNOS activation in PHT gastric mucosa and the role of TNF-alpha in this signaling remain unknown. In PHT gastric mucosa we studied (1) eNOS phosphorylation (at serine 1177) required for its activation; (2) association of the phosphatidylinositol 3-kinase (PI 3-kinase), and its downstream effector Akt, with eNOS; and, (3) whether TNF-alpha neutralization affects eNOS phosphorylation and PI 3-kinase-Akt activation. To determine human relevance, we used human microvascular endothelial cells to examine directly whether TNF-alpha stimulates eNOS phosphorylation via PI 3-kinase. PHT gastric mucosa has significantly increased (1) eNOS phosphorylation at serine 1177 by 90% (P <.01); (2) membrane translocation (P <.05) and phosphorylation (P <.05) of p85 (regulatory subunit of PI 3-kinase) by 61% and 85%, respectively; (3) phosphorylation (P <.01) and activity (P <.01) of Akt by 40% and 52%, respectively; and (4) binding of Akt to eNOS by as much as 410% (P <.001). Neutralizing anti-TNF-alpha antibody significantly reduced p85 phosphorylation, phosphorylation and activity of Akt, and eNOS phosphorylation in PHT gastric mucosa to normal levels. Furthermore, TNF-alpha stimulated eNOS phosphorylation in human microvascular endothelial cells. In conclusion, these findings show that in PHT gastric mucosa, TNF-alpha stimulates eNOS phosphorylation at serine 1177 (required for its activation) via the PI 3-kinase-Akt signal transduction pathway.     

B-cell adaptor for PI3K (BCAP) negatively regulates Toll-like receptor signaling through activation of PI3K

Toll-like receptors (TLRs) recognize pathogens and their components, thereby initiating immune responses to infectious organisms. TLR ligation leads to the activation of NF-κB and MAPKs through well-defined pathways, but it has remained unclear how TLR signaling activates PI3K, which provides an inhibitory pathway limiting TLR responses. Here, we show that the signaling adapter B-cell adaptor for PI3K (BCAP) links TLR signaling to PI3K activation. BCAP-deficient macrophages and mice are hyperresponsive to TLR agonists and have reduced PI3K activation. The ability of BCAP to inhibit TLR responses requires its capacity to bind PI3K. BCAP is constitutively phosphorylated and associated with the p85 subunit of PI3K in macrophages. This tyrosine-phosphorylated BCAP is transiently enriched in the membrane fraction in response to LPS treatment, suggesting a model whereby TLR signaling causes the phosphorylation of the small amount of BCAP that is associated with membranes in the resting state or the translocation of phosphorylated BCAP from the cytoplasm to the membrane. This accumulation of tyrosine-phosphorylated BCAP at the membrane with its associated PI3K would then allow for the catalysis of Ptd Ins P2 to Ptd Ins P3 and downstream PI3K-dependent signals. Therefore, BCAP is an essential activator of the PI3K pathway downstream of TLR signaling, providing a brake to limit potentially pathogenic excessive TLR responses.     

Phage display selection of ligand residues important for Src homology 3 domain binding specificity.

The Src homology 3 (SH3) domain is a 50-aa modular unit present in many cellular proteins involved in intracellular signal transduction. It functions to direct protein-protein interactions through the recognition of proline-rich motifs on associated proteins. SH3 domains are important regulatory elements that have been demonstrated to specify distinct regulatory pathways important for cell growth, migration, differentiation, and responses to the external milieu. By the use of synthetic peptides, ligands have been shown to consist of a minimum core sequence and to bind to SH3 domains in one of two pseudosymmetrical orientations, class I and class II. The class I sites have the consensus sequence ZP(L/P)PP psi P whereas the class II consensus is PP psi PPZ (where psi is a hydrophobic residue and Z is a SH3 domain-specific residue). We previously showed by M13 phage display that the Src, Fyn, Lyn, and phosphatidylinositol 3-kinase (PI3K) SH3 domains preferred the same class I-type core binding sequence, RPLPP psi P. These results failed to explain the specificity for cellular proteins displayed by SH3 domains in cells. In the current study, class I and class II core ligand sequences were displayed on the surface of bacteriophage M13 with five random residues placed either N- or C-terminal of core ligand residues. These libraries were screened for binding to the Src, Fyn, Lyn, Yes, and PI3K SH3 domains. By this approach, additional ligand residue preferences were identified that can increase the affinity of SH3 peptide ligands at least 20-fold compared with core peptides. The amino acids selected in the flanking sequences were similar for Src, Fyn, and Yes SH3 domains; however, Lyn and PI3K SH3 domains showed distinct binding specificities. These results indicate that residues that flank the core binding sequences shared by many SH3 domains are important determinants of SH3 binding affinity and selectivity.     

MAP kinases and mTOR mediate insulin-induced phosphorylation of Insulin Receptor Substrate-1 on serine residues 307, 612 and 632

Aim/hypothesis Insulin-induced IRS-1 serine phosphorylation could be physiologically important to regulate insulin action. In a hyperinsulinaemic state such as obesity or Type 2 diabetes, this phosphorylation could be modified and exacerbate insulin resistance. We aimed at identifying serine residues in IRS-1 phosphorylated in response to insulin stimulation and at determining the involved kinases.Methods 3T3-L1 adipocytes, muscle and adipose tissue of mice were subjected to Western Blot analysis with phosphospecific antibodies to identify phosphorylation sites in IRS-1 following insulin treatment. Pharmacological inhibitors were used to determine the serine kinases involved in this phosphorylation.Results In 3T3-L1 adipocytes, insulin promoted the phosphorylation of serine 307, 612 and 632 with Serine^612/632 more rapidly phosphorylated than Serine³⁰⁷. Insulin-induced phosphorylation of Serine³⁰⁷ was dependent on the activation of a PI 3-kinase/mTOR pathway. The phosphorylation of Serine^612/632 required the activation of the MAP kinase pathway following short-term insulin stimulation and activation of the PI 3-kinase/mTOR pathway following prolonged insulin stimulation. Phosphorylation of Serine³⁰⁷ and Serine⁶³² occurred in vivo in skeletal muscle and white adipose tissue of mice injected with insulin and was dependent on the activation of mTOR. Moreover, inhibition of mTOR led to a persistent PI 3-kinase activation by insulin.Conclusion/Interpretation Insulin-induced IRS-1 serine phosphorylation is a complex process involving different sites and kinases. This complexity could be physiologically important to accurately regulate insulin signalling. Abnormal phosphorylation of these serine residues in hyperinsulinaemic state could participate in the down-regulation of insulin signalling.Abbreviations PI 3-kinase phosphatidylinositol 3-kinase - mTOR mammalian target of rapamycin - APS adaptor with a PH and SH2 domains - Shc Src Homology Collagen - SH2 Src Homology 2 - PTB phosphotyrosine binding - MAP mitogen-activated protein - MEK mitogen-activated protein kinase kinase - PKB protein kinase B - PDGF platelet derived growth factor - JNK c-Jun NH2 terminal kinase - PMA phorbol myristate acetate - PIP3 phosphatidylinositol 3,4,5 triphosphates     

HCV蛋白NS5A对PI3K/Akt通路的分子调节作用

目的研究丙肝病毒（HCV）蛋白NS5A对PI3K/Akt信号的调节机制及其意义。方法 HepG2细胞分别转染NS5A质粒和对照载体。提取总蛋白,用Western blotting法分析PI3K信号Akt磷酸化水平,并用免疫沉淀法检测p85酪氨酸磷酸化水平及p85与p110蛋白间的相互作用。结果 NS5A转染细胞p-Akt蛋白水平上调,同时p85酪氨酸磷酸化水平显著提高,但催化亚基p110与调节亚基p85的结合作用没有明显变化。结论丙肝病毒（HCV）蛋白NS5A可以和PI3Kp85亚基结合而调节PI3K/Akt信号通路,但其机制可能有p85/p110以外的机制。这可能为临床丙肝IFN敏感性的诊断与治疗提供依据。     

Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein

Seasonal epidemics and periodic worldwide pandemics caused by influenza A viruses are of continuous concern. The viral nonstructural (NS1) protein is a multifunctional virulence factor that antagonizes several host innate immune defenses during infection. NS1 also directly stimulates class IA phosphoinositide 3-kinase (PI3K) signaling, an essential cell survival pathway commonly mutated in human cancers. Here, we present a 2.3-Å resolution crystal structure of the NS1 effector domain in complex with the inter-SH2 (coiled-coil) domain of p85β, a regulatory subunit of PI3K. Our data emphasize the remarkable isoform specificity of this interaction, and provide insights into the mechanism by which NS1 activates the PI3K (p85β:p110) holoenzyme. A model of the NS1:PI3K heterotrimeric complex reveals that NS1 uses the coiled-coil as a structural tether to sterically prevent normal inhibitory contacts between the N-terminal SH2 domain of p85β and the p110 catalytic subunit. Furthermore, in this model, NS1 makes extensive contacts with the C2/kinase domains of p110, and a small acidic α-helix of NS1 sits adjacent to the highly basic activation loop of the enzyme. During infection, a recombinant influenza A virus expressing NS1 with charge-disruption mutations in this acidic α-helix is unable to stimulate the production of phosphatidylinositol 3,4,5-trisphosphate or the phosphorylation of Akt. Despite this, the charge-disruption mutations in NS1 do not affect its ability to interact with the p85β inter-SH2 domain in vitro. Overall, these data suggest that both direct binding of NS1 to p85β (resulting in repositioning of the N-terminal SH2 domain) and possible NS1:p110 contacts contribute to PI3K activation.     

Phosphoinositide-3 kinase binds to a proline-rich motif in the Na+, K+-ATPase alpha subunit and regulates its trafficking

Endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I(A) phosphoinositide-3 kinase (PI3K-I(A)) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I(A), through its p85alpha subunit-SH3 domain, binds to a proline-rich region in the Na(+),K(+)-ATPase catalytic alpha subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na(+),K(+)-ATPase alpha subunit and results in increased PI3K-I(A) activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na(+),K(+)-ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I(A) and its activation during Na(+),K(+)-ATPase endocytosis in response to G protein-coupled receptor signals.     

v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation

v-Crk induces cellular tyrosine phosphorylation and transformation of chicken embryo fibroblasts (CEF). We studied the molecular mechanism of the v-Crk-induced transformation. Experiments with Src homology (SH)2 and SH3 domain mutants revealed that the induction of tyrosine phosphorylation of cellular proteins requires only the SH2 domain, but both the SH2 and SH3 domains are required for complete transformation. Analysis of three well defined signaling pathways, the mitogen-activated protein kinase (MAPK) pathway, the Jun N-terminal kinase (JNK) pathway, and the phosphoinositide 3-kinase (PI3K)/AKT pathway, demonstrated that only the PI3K/AKT pathway is constitutively activated in v-Crk-transformed CEF. Both the SH2 and SH3 domains are required for this activation of the PI3K/AKT pathway in CEF. We also found that the colony formation of CEF is strongly induced by a constitutively active PI3K mutant, and that a PI3K inhibitor, LY294002, suppresses the v-Crk-induced transformation. These results strongly suggest that constitutive activation of the PI3K/AKT pathway plays an essential role in v-Crk-induced transformation of CEF.     

Growth factor-stimulated phosphorylation of Akt and p70(S6K) is differentially inhibited by LY294002 and Wortmannin

The phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 (LY) and wortmannin (WM), are widely used to examine the role of PI3K in growth factor signaling. These compounds inhibit the kinase action of PI3K, thus preventing the accumulation of PI(3,4,5)P3 and PI(3,4)P2 (PIs) and subsequent phosphorylation and activation of the downstream effectors of PI3K, Akt and p70(S6K). The efficacy of these inhibitors has been demonstrated by measuring cellular levels of PIs or the kinase activity of immunoprecipitated PI3K. However, their effects on activation of Akt and p70(S6K), more widely used markers of PI3K activation, has not been formally tested. We have examined the effects of LY and WM on phosphorylation of Akt and p70(S6K) by insulin-like growth factor-I, insulin, and platelet-derived growth factor in skeletal muscle cells. LY is much less effective in blocking the phosphorylation of Akt than p70(S6K); at concentrations which completely inhibit phosphorylation of p70(S6K), phosphorylation of Akt is only partially inhibited by LY. WM also inhibits IGF-I-stimulated phosphorylation of Akt and p70(S6K) with unequal potency but is equally effective in blocking insulin-stimulated phosphorylation of these peptides. Our data demonstrate that inhibiting PI3K signaling through one of its downstream mediators (p70(S6K)) may not indicate complete blockage of the PI3K pathway which may be signaling through an alternate downstream branch (Akt). These findings indicate that the efficacy of LY and WM in blocking PI3K-activation of Akt and p70(S6K) must be tested within the context of every experiment, and that the results obtained with the use of these inhibitors must be interpreted according to their specific effects on the PI3K/Akt and PI3K/p70(S6K) signaling pathways.     

p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.     

Fatty acid-induced insulin resistance: decreased muscle PI3K activation but unchanged Akt phosphorylation.

The mechanisms by which elevated plasma nonesterified fatty acid (NEFA) levels induce skeletal muscle insulin resistance remain unclear. A NEFA-induced defect in the activation of PI3K, which plays a key role in insulin's stimulation of glucose transport, has been invoked. We sought to examine the effects of elevated plasma NEFA (approximately 1 mmol/liter) on muscle PI3K activity, insulin receptor substrate (IRS)-1 (important for activation of PI3K), and Akt, which is downstream of PI3K and activated by phosphorylation on serine and threonine in a PI3K-dependent manner. Ten normal men [age, 37 +/- 9 yr (mean +/- SD); body mass index, 25.2 +/- 3.8 kg/m(2)] underwent two 5-h hyperinsulinemic (80 mU/m(2) x min) euglycemic clamps with basal and end of clamp biopsies of the vastus lateralis muscle. Plasma NEFAs were increased in one study by infusion of 20% Intralipid (1 ml/min) and heparin (900 U/h) throughout and for 2.5 h beforehand. Skeletal muscle protein levels were quantified by Western blotting. Elevated plasma NEFA reduced whole-body insulin-stimulated glucose disposal by 24% (42.1 +/- 4.0 vs. 54.8 +/- 3.6 micromol/kg x min; P < 0.001). Basal muscle IRS-1 was the same in the two studies. IRS-1 levels decreased by 40% in the control glucose clamps (P < 0.005), but did not change during the Intralipid study. Total tyrosine phosphorylated IRS-1 increased by 29% during the control clamps (P < 0.05), but by only 18% (NS) during the Intralipid studies. Total levels of p85alpha subunit of PI3K and Akt were not influenced by plasma NEFA levels either in the basal state or during the glucose clamps. The insulin-induced increase in IRS-1-associated PI3K activity was impaired by elevated NEFA, so that activity at the end of the clamps with Intralipid was 35% lower than in the control clamps (P < 0.05). The percentage reduction in PI3K activation correlated with the reduction in insulin-stimulated glucose disappearance rate that was induced by elevated NEFA (r = 0.70; P < 0.05). Basal P-ser- and P-thr-Akt levels were very low and unaffected by NEFA levels. The glucose clamps resulted in a marked increase in P-ser and P-thr Akt levels. Despite the decrease in PI3K in the Intralipid study, no defect in Akt phosphorylation was found. In summary, NEFA-induced insulin resistance is associated with an impairment of IRS-1 tyrosine phosphorylation and IRS-1-associated PI3K activation. Down-regulation of IRS-1 levels is also impaired. The NEFA-induced defect in muscle glucose uptake appears to be a consequence of a defect in the insulin-signaling pathway leading to impaired PI3K activation. This in turn may lead to impaired glucose transport through an Akt-independent pathway because Akt phosphorylation was unaffected by elevated NEFA levels.     

Persistent activation of phosphatidylinositol 3-kinase causes insulin resistance due to accelerated insulin-induced insulin receptor substrate-1 degradation in 3T3-L1 adipocytes

Recently, we have reported that the overexpression of a membrane-targeted phosphatidylinositol (PI) 3-kinase (p110CAAX) stimulated p70S6 kinase, Akt, glucose transport, and Ras activation in the absence of insulin but inhibited insulin-stimulated glycogen synthase activation and MAP kinase phosphorylation in 3T3-L1 adipocytes. To investigate the mechanism of p110CAAX-induced cellular insulin resistance, we have now studied the effect of p110CAAX on insulin receptor substrate (IRS)-1 protein. Overexpression of p110CAAX alone decreased IRS-1 protein levels to 63+/-10% of control values. Insulin treatment led to an IRS-1 gel mobility shift (most likely caused by serine/threonine phosphorylation), with subsequent IRS-1 degradation. Moreover, insulin-induced IRS-1 degradation was enhanced by expression of p110CAAX (61+/-16% vs. 13+/-15% at 20 min, and 80+/-8% vs. 41+/-12% at 60 min, after insulin stimulation with or without p110CAAX expression, respectively). In accordance with the decreased IRS-1 protein, the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase was also decreased in the p110CAAX-expressing cells, and IRS-1-associated PI 3-kinase activity was decreased despite the fact that total PI 3-kinase activity was increased. Five hours of wortmannin pretreatment inhibited both serine/threonine phosphorylation and degradation of IRS-1 protein. These results indicate that insulin treatment leads to serine/threonine phosphorylation of IRS-1, with subsequent IRS-1 degradation, through a PI 3-kinase-sensitive mechanism. Consistent with this, activated PI 3-kinase phosphorylates IRS-1 on serine/threonine residues, leading to IRS- 1 degradation. The similar finding was observed in IRS-2 as well as IRS-1. These results may also explain the cellular insulin-resistant state induced by chronic p110CAAX expression.     

PKCα Activation of p120-Catenin Serine 879 Phospho-Switch Disassembles VE-Cadherin Junctions and Disrupts Vascular Integrity

Rationale: Adherens junctions (AJs) are the primary intercellular junctions in microvessels responsible for endothelial barrier function. Homophilic adhesion of vascular endothelial (VE) cadherin forms AJs, which are stabilized by binding of p120-catenin (p120). p120 dissociation from VE-cadherin results in loss of VE-cadherin homotypic interaction and AJ disassembly; however, the signaling mechanisms regulating p120 dissociation from VE-cadherin are not understood. Objective: To address the mechanism of protein kinase C (PKC)-α function in increasing endothelial permeability, we determined the role of PKCα phosphorylation of p120 in mediating disruption of AJ integrity. Methods and Results: We showed that PKCα phosphorylation of p120 at serine (S)879 in response to thrombin or lipopolysaccharide challenge reduced p120 binding affinity for VE-cadherin and mediated AJ disassembly secondary to VE-cadherin internalization. In studies in mouse lung vessels, expression of the phosphodeficient S879A-p120 mutant prevented the increase in vascular permeability induced by activation of the thrombin receptor PAR-1. Conclusions: PKCα phosphorylation of p120 at S879 is a critical phospho-switch mediating disassociation of p120 from VE-cadherin that results in AJ disassembly. Therefore, blocking PKCα-mediated p120 phosphorylation represents a novel targeted anti-inflammatory strategy to prevent disruption of vascular endothelial barrier function.     

Association of the p85 regulatory subunit of phosphatidylinositol 3- kinase with an essential erythropoietin receptor subdomain

Using an active, HAI epitope-tagged form of the murine erythropoietin (EPO) receptor and via direct coimmunoprecipitation, the p85 regulatory subunit of phosphatidyl inositol-3 kinase (p85/PI3-K) is shown to associate with the EPO receptor in transfected FDC-P1 cell lines. Coimmunoprecipitation of p85 with epitope-tagged EPO receptors was observed initially in FDC-HER cells labeled metabolically with [32P]orthophosphate, and association of these factors was confirmed by Western analyses of receptor immunoprecipitates using p85 antiserum. Interestingly, this association occurred in the absence of ligand, and exposure of FDC-HER cells to EPO did not detectably affect levels of receptor-associated p85 or overall levels of p85 phosphorylation. However, EPO was observed to stimulated the rapid formation of phosphatidylinositol 32P-phosphate in FDC-HER and FDC-ER cells. Through baculovirus-mediated expression of epitope-tagged EPO receptor forms in SF9 cells, domains for p85 association were mapped. Analyses of receptor forms with cytosolic truncations and deletions delineated a candidate subdomain for p85 binding to an essential extended box-2 region (P329-E374; including a putative motif for SH2 binding, Y343LVL). These findings extend a mechanistic alignment between the EPO receptor and protein tyrosine kinase-encoding receptors that likewise activate PI3-K, and expand the importance of further defining pathways to PI3-K activation.