scholarly journals Molecular Mechanism of Insulin-Induced Degradation of Insulin Receptor Substrate 1

2002 ◽  
Vol 22 (4) ◽  
pp. 1016-1026 ◽  
Author(s):  
Rachel Zhande ◽  
John J. Mitchell ◽  
Jiong Wu ◽  
Xiao Jian Sun
Keyword(s):  
Insulin Receptor ◽  
Molecular Mechanism ◽  
Degradation Pathway ◽  
In Vivo Studies ◽  
Temperature Sensitive ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
Proteasome Degradation ◽  
Ubiquitin Proteasome

ABSTRACT Insulin receptor substrate 1 (IRS-1) plays an important role in the insulin signaling cascade. In vitro and in vivo studies from many investigators have suggested that lowering of IRS-1 cellular levels may be a mechanism of disordered insulin action (so-called insulin resistance). We previously reported that the protein levels of IRS-1 were selectively regulated by a proteasome degradation pathway in CHO/IR/IRS-1 cells and 3T3-L1 adipocytes during prolonged insulin exposure, whereas IRS-2 was unaffected. We have now studied the signaling events that are involved in activation of the IRS-1 proteasome degradation pathway. Additionally, we have addressed structural elements in IRS-1 versus IRS-2 that are required for its specific proteasome degradation. Using ts20 cells, which express a temperature-sensitive mutant of ubiquitin-activating enzyme E1, ubiquitination of IRS-1 was shown to be a prerequisite for insulin-induced IRS-1 proteasome degradation. Using IRS-1/IRS-2 chimeric proteins, the N-terminal region of IRS-1 including the PH and PTB domains was identified as essential for targeting IRS-1 to the ubiquitin-proteasome degradation pathway. Activation of phosphatidylinositol 3-kinase is necessary but not sufficient for activating and sustaining the IRS-1 ubiquitin-proteasome degradation pathway. In contrast, activation of mTOR is not required for IRS-1 degradation in CHO/IR cells. Thus, our data provide insight into the molecular mechanism of insulin-induced activation of the IRS-1 ubiquitin-proteasome degradation pathway.

14-3-3 Facilitates Insulin-Stimulated Intracellular Trafficking of Insulin Receptor Substrate 1

2002 ◽  
Vol 16 (3) ◽  
pp. 552-562 ◽  
Author(s):  
Xiaoqin Xiang ◽  
Mingsheng Yuan ◽  
Ying Song ◽  
Neil Ruderman ◽  
Rong Wen ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Intracellular Trafficking ◽  
High Speed ◽  
Insulin Treatment ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
Integral Role ◽  
Endogenous Proteins

Abstract The appearance of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and PI3K in a high-speed pellet fraction (HSP) is thought to be a key event in insulin action. Conversely, the disappearance of the IRS-1/PI3K complex from this fraction has been linked to insulin desensitization. The present study examines the role of 14-3-3, a specific phospho-serine binding protein, in mediating the disappearance of IRS-1 from the HSP after insulin treatment. An in vitro pull-down assay using recombinant 14-3-3 revealed that insulin enhances the association of 14-3-3 with IRS-1 in cultured adipocytes and that this is completely inhibited by wortmannin. An association of IRS-1 and 14-3-3 was also observed and was maximal after stimulation by insulin, when endogenous proteins were immunoprecipitated. Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate, and okadaic acid, other agents that cause serine/threonine phosphorylation of IRS-1, also stimulated IRS binding to 14-3-3. The enhancement of IRS-1 binding to 14-3-3 by insulin was accompanied by movement of IRS-1 and the p85 subunit of PI3K from the HSP to the cytosol. In keeping with a key role of 14-3-3 in mediating this redistribution of IRS-1, the complexes of IRS-1 and 14-3-3 were found in the cytosol but not in the HSP of insulin-treated cells. In addition, colocalization of IRS-1 and 14-3-3 was observed in the cytoplasm after insulin treatment by confocal microscopy. Finally, the addition of a phosphorylated 14-3-3 binding peptide to an adipocyte homogenate (to remove 14-3-3 from IRS-1) increased the abundance of IRS-1/PI3K complexes in the HSP and decreased their abundance in the cytosol. These findings strongly suggest that 14-3-3 participates in the intracellular trafficking of IRS-1 by promoting the displacement of serine-phosphorylated IRS-1 from particular structures. They also suggest that 14-3-3 proteins could play an integral role in the process of insulin desensitization.

scholarly journals Glycogen synthase kinase 3β mediates high glucose-induced ubiquitination and proteasome degradation of insulin receptor substrate 1

2010 ◽  
Vol 206 (2) ◽  
pp. 171-181 ◽  
Author(s):  
Sanhua Leng ◽  
Wenshuo Zhang ◽  
Yanbin Zheng ◽  
Ziva Liberman ◽  
Christopher J Rhodes ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Molecular Mechanism ◽  
High Glucose ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Insulin Receptor Substrate ◽  
Glycogen Synthase Kinase 3Β ◽  
Proteasome Degradation ◽  
Protein Levels

High glucose (HG) has been shown to induce insulin resistance in both type 1 and type 2 diabetes. However, the molecular mechanism behind this phenomenon is unknown. Insulin receptor substrate (IRS) proteins are the key signaling molecules that mediate insulin's intracellular actions. Genetic and biological studies have shown that reductions in IRS1 and/or IRS2 protein levels are associated with insulin resistance. In this study we have shown that proteasome degradation of IRS1, but not of IRS2, is involved in HG-induced insulin resistance in Chinese hamster ovary (CHO) cells as well as in primary hepatocytes. To further investigate the molecular mechanism by which HG induces insulin resistance, we examined various molecular candidates with respect to their involvement in the reduction in IRS1 protein levels. In contrast to the insulin-induced degradation of IRS1, HG-induced degradation of IRS1 did not require IR signaling or phosphatidylinositol 3-kinase/Akt activity. We have identified glycogen synthase kinase 3β (GSK3β or GSK3B as listed in the MGI Database) as a kinase required for HG-induced serine332 phosphorylation, ubiquitination, and degradation of IRS1. Overexpression of IRS1 with mutation of serine332 to alanine partially prevents HG-induced IRS1 degradation. Furthermore, overexpression of constitutively active GSK3β was sufficient to induce IRS1 degradation. Our data reveal the molecular mechanism of HG-induced insulin resistance, and support the notion that activation of GSK3β contributes to the induction of insulin resistance via phosphorylation of IRS1, triggering the ubiquitination and degradation of IRS1.

Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase CβII in the diabetic fat tissue

2008 ◽  
Vol 294 (6) ◽  
pp. E1169-E1177 ◽  
Author(s):  
Ziva Liberman ◽  
Batya Plotkin ◽  
Tamar Tennenbaum ◽  
Hagit Eldar-Finkelman
Keyword(s):  
Protein Kinase ◽  
Insulin Receptor ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Insulin Receptor Substrate ◽  
Fat Tissue ◽  
Insulin Receptor Substrate 1 ◽  
Gsk 3Β

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is an important negative modulator of insulin signaling. Previously, we showed that glycogen synthase kinase-3 (GSK-3) phosphorylates IRS-1 at Ser332. However, the fact that GSK-3 requires prephosphorylation of its substrates suggested that Ser336 on IRS-1 was the “priming” site phosphorylated by an as yet unknown protein kinase. Here, we sought to identify this “priming kinase” and to examine the phosphorylation of IRS-1 at Ser336 and Ser332 in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser336. Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKCα or PKCβII isoforms in cells enhanced IRS-1 phosphorylation at Ser336 and Ser332, and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser336. The expression level and activation state of PKCβII, but not PKCα, were remarkably elevated in the fat tissues of diabetic ob/ob mice and in high-fat diet-fed mice compared with that from lean animals. Elevated levels of PKCβII were also associated with enhanced phosphorylation of IRS-1 at Ser336/332 and elevated activity of GSK-3β. Finally, adenoviral mediated expression of PKCβII in adipocytes enhancedphosphorylation of IRS-1 at Ser336. Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCβII and GSK-3 at Ser336 and Ser332. Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.

Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression

1994 ◽  
Vol 14 (11) ◽  
pp. 7466-7475
Author(s):  
B H Jhun ◽  
D W Rose ◽  
B L Seely ◽  
L Rameh ◽  
L Cantley ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Dna Synthesis ◽  
Insulin Receptor ◽  
Signal Transduction Pathway ◽  
Sh2 Domain ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Substrate 1 ◽  
Phosphatidylinositol 3

We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction.

scholarly journals Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression.

1994 ◽  
Vol 14 (11) ◽  
pp. 7466-7475 ◽  
Author(s):  
B H Jhun ◽  
D W Rose ◽  
B L Seely ◽  
L Rameh ◽  
L Cantley ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Dna Synthesis ◽  
Insulin Receptor ◽  
Signal Transduction Pathway ◽  
Sh2 Domain ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Substrate 1 ◽  
Phosphatidylinositol 3

We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction.

scholarly journals Modulation of human insulin receptor substrate-1 tyrosine phosphorylation by protein kinase Cdelta

2004 ◽  
Vol 378 (1) ◽  
pp. 105-116 ◽  
Author(s):  
Michael W. GREENE ◽  
Nick MORRICE ◽  
Robert S. GAROFALO ◽  
Richard A. ROTH
Keyword(s):  
Fatty Acid ◽  
Protein Kinase ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Inhibitory Effect ◽  
Insulin Receptor Substrate ◽  
Triple Mutant ◽  
Insulin Receptor Substrate 1

Non-esterified fatty acid (free fatty acid)-induced activation of the novel PKC (protein kinase C) isoenzymes PKCδ and PKCθ correlates with insulin resistance, including decreased insulin-stimulated IRS-1 (insulin receptor substrate-1) tyrosine phosphorylation and phosphoinositide 3-kinase activation, although the mechanism(s) for this resistance is not known. In the present study, we have explored the possibility of a novel PKC, PKCδ, to modulate directly the ability of the insulin receptor kinase to tyrosine-phosphorylate IRS-1. We have found that expression of either constitutively active PKCδ or wild-type PKCδ followed by phorbol ester activation both inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in vivo. Activated PKCδ was also found to inhibit the IRS-1 tyrosine phosphorylation in vitro by purified insulin receptor using recombinant full-length human IRS-1 and a partial IRS-1–glutathione S-transferase-fusion protein as substrates. This inhibition in vitro was not observed with a non-IRS-1 substrate, indicating that it was not the result of a general decrease in the intrinsic kinase activity of the receptor. Consistent with the hypothesis that PKCδ acts directly on IRS-1, we show that IRS-1 can be phosphorylated by PKCδ on at least 18 sites. The importance of three of the PKCδ phosphorylation sites in IRS-1 was shown in vitro by a 75–80% decrease in the incorporation of phosphate into an IRS-1 triple mutant in which Ser-307, Ser-323 and Ser-574 were replaced by Ala. More importantly, the mutation of these three sites completely abrogated the inhibitory effect of PKCδ on IRS-1 tyrosine phosphorylation in vitro. These results indicate that PKCδ modulates the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by direct phosphorylation of the IRS-1 molecule.

scholarly journals Role of Substance P in the Regulation of Glucose Metabolism via Insulin Signaling-Associated Pathways

Endocrinology ◽  
2011 ◽  
Vol 152 (12) ◽  
pp. 4571-4580 ◽  
Author(s):  
Iordanes Karagiannides ◽  
Kyriaki Bakirtzi ◽  
Efi Kokkotou ◽  
Dimitris Stavrakis ◽  
Kara Gross Margolis ◽  
...  
Keyword(s):  
Weight Gain ◽  
Protein Kinase ◽  
Glucose Metabolism ◽  
Substance P ◽  
Insulin Receptor ◽  
Blood Levels ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
Human Preadipocytes

Substance P (SP), encoded by the tachykinin 1 (Tac1) gene, is the most potent tachykinin ligand for the high-affinity neurokinin-1 receptor (NK-1R). We previously reported that NK-1R-deficient mice show less weight gain and reduced circulating levels of leptin and insulin in response to a high-fat diet (HFD) and demonstrated the presence of functional NK-1R in isolated human preadipocytes. Here we assessed the effects of SP on weight gain in response to HFD and determined glucose metabolism in Tac1-deficient (Tac1−/−) mice. The effect of SP on the expression of molecules that may predispose to reduced glucose uptake was also determined in isolated human mesenteric, omental, and sc preadipocytes. We show that although weight accumulation in response to HFD was similar between Tac1−/− mice and wild-type littermates, Tac1−/− mice demonstrated lower glucose and leptin and increased adiponectin blood levels and showed improved responses to insulin challenge after HFD. SP stimulated phosphorylation of c-Jun N-terminal kinase, protein kinase Cθ, mammalian target of rapamycin, and inhibitory serine insulin receptor substrate-1 phosphorylation in human preadipocytes in vitro. Preincubation of human mesenteric preadipocytes with the protein kinase Cθ pseudosubstrate inhibitor reduced insulin receptor substrate 1 phosphorylation in response to SP. Lastly, SP also induced insulin receptor substrate-1 phosphorylation in mature human sc adipocytes. Our results demonstrate an important role for SP in adipose tissue responses and obesity-associated pathologies. These novel SP effects on molecules that enhance insulin resistance at the adipocyte level may reflect an important role for this peptide in the pathophysiology of type 2 diabetes.

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