Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study the role of kinase dimerization in the activation of the insulin receptor (IR) and the insulin-like growth factor receptor-1 (IGF-1R), we have cloned, expressed, and purified monomeric and dimeric forms of the corresponding soluble kinase domains via the baculovirus expression system. Dimerization of the kinases was achieved by fusion of the kinase domains to the homodimeric glutathione S-transferase (GST). Kinetic analyses revealed that kinase dimerization results in substantial increases (10-100-fold) in the phosphotransferase activity in both the auto- and substrate phosphorylation reactions. Furthermore, kinase dimerization rendered the autophosphorylation reaction concentration-independent. However, whereas dimerization was required for the rapid autophosphorylation of the kinases, it was not essential for the enhanced kinase activity in substrate phosphorylation reactions. Comparison of HPLC-phosphopeptide maps of the monomeric and dimeric kinases revealed that dimerization leads to an increased phosphorylation of the regulatory activation loop of the kinases, strongly suggesting that bis- and trisphosphorylation of the activation loop are mediated by transphosphorylation within the kinase dimers. Most strikingly, limited proteolysis revealed that GST-mediated dimerization by itself had a major impact on the conformation of the activation loop by stabilizing a conformation that corresponds to the active, phosphorylated form of the kinase. Thus, in analogy to the insulin/IGF-1-ligated holoreceptors, the dimeric GST-kinases are primed to rapid autophosphorylation by an increase in the local concentration of both phosphoryl donor and phosphoryl acceptor sites and by a dimerization-induced conformational change of the activation loop that leads to an efficient transphosphorylation of the regulatory tyrosine residues.
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PMID:Dimerization-induced activation of soluble insulin/IGF-1 receptor kinases: an alternative mechanism of activation. 1171 81

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) that translocates from the nucleus to the cytoplasm and that Tyr-phosphorylated Sam68 associates with the SH2 domains of p85 PI3K and GAP, in vivo and in vitro. In the present work, we have further demonstrated the cytoplasmic localization of Sam68, which is increased in cells overexpressing IR. Besides, we sought to further study the association of Sam68 with the Ras-GAP pathway by assessing the interactions with SH3 domains of Grb2. We employed GST-fusion proteins containing the SH3 domains of Grb2 (N or C), and recombinant Sam68 for in vitro studies. In vivo studies of protein-protein interaction were assessed by co-immunoprecipitation experiments with specific antibodies against Sam68, GAP, Grb2, SOS, and phosphotyrosine; and by affinity precipitation with the fusion proteins (SH3-Grb2). Insulin stimulation of HTC-IR cells promotes phosphorylation of Sam68 and its association with the SH2 domains of GAP. Sam68 is constitutively associated with the SH3 domains of Grb2 and it does not change upon insulin stimulation, but Sam68 is Tyr-phosphorylated and promotes the association of GAP with the Grb2-SOS complex. In vitro studies with fusion proteins showed that Sam68 association with Grb2 is preferentially mediated by the C-terminal SH3 domains of Grb2. In conclusion, Sam68 is a substrate of the IR and may have a role as a docking protein in IR signaling, recruiting GAP to the Grb2-SOS complex, and in this way it may modulate Ras activity.
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PMID:Sam68 associates with the SH3 domains of Grb2 recruiting GAP to the Grb2-SOS complex in insulin receptor signaling. 1211 20

SHIP2 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains motifs susceptible to mediate protein-protein interaction. Using yeast two-hybrid, GST-pulldown, and coimmunoprecipitation studies, we isolated the CAP cDNA as a specific partner of SHIP2 proline-rich domain and showed by GST-pulldown experiments that the interaction took place with the SH3C of CAP. The interaction was not modulated in COS-7 cells stimulated by EGF neither in CHO cells overexpressing the insulin receptor in the presence or absence of insulin stimulation. We also showed that SHIP2 was able to coimmunoprecipitate with endogenous c-Cbl protein in the absence of CAP and with the insulin receptor in CHO-IR cell extracts. The presence of SHIP2 in a complex around the insulin receptor could account for the very specific increase in insulin sensitivity of SHIP2 knock-out mice.
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PMID:The c-Cbl-associated protein and c-Cbl are two new partners of the SH2-containing inositol polyphosphate 5-phosphatase SHIP2. 1250 11

Non-esterified fatty acid (free fatty acid)-induced activation of the novel PKC (protein kinase C) isoenzymes PKCdelta and PKCtheta 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, PKCdelta, to modulate directly the ability of the insulin receptor kinase to tyrosine-phosphorylate IRS-1. We have found that expression of either constitutively active PKCdelta or wild-type PKCdelta followed by phorbol ester activation both inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in vivo. Activated PKCdelta 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 PKCdelta acts directly on IRS-1, we show that IRS-1 can be phosphorylated by PKCdelta on at least 18 sites. The importance of three of the PKCdelta 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 PKCdelta on IRS-1 tyrosine phosphorylation in vitro. These results indicate that PKCdelta modulates the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by direct phosphorylation of the IRS-1 molecule.
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PMID:Modulation of human insulin receptor substrate-1 tyrosine phosphorylation by protein kinase Cdelta. 1458 92

In the present study, we tested the hypothesis that 17beta-estradiol (betaE2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H(2)O(2))-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both betaE2 and 17alpha-estradiol (alphaE2) significantly protected against H(2)O(2)-induced retinal neuron degeneration; however, progesterone had no effect. betaE2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [(32)gammaATP] were used as substrate. Phospho-Akt levels were also transiently increased by betaE2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of betaE2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of betaE2, suggesting that betaE2 mediates its effect through some PI3K-dependent pathway, independent of the estrogen receptor. Pull-down experiments with glutathione S-transferase fused to the N-Src homology 2 domain of p85, the regulatory subunit of PI3K, indicated that betaE2 and alphaE2, but not progesterone, identified phosphorylated insulin receptor beta-subunit (IRbeta) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IRbeta activation of PI3K. Systemic administration of betaE2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of betaE2 activated retinal IRbeta, but not the insulin-like growth factor receptor-1, and produced a transient increase in PI3K activity and phosphorylation of Akt in rat retinas. The results show that estrogen has retinal neuroprotective properties in vivo and in vitro and suggest that the insulin receptor/PI3K/Akt signaling pathway is involved in estrogen-mediated retinal neuroprotection.
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PMID:Involvement of insulin/phosphoinositide 3-kinase/Akt signal pathway in 17 beta-estradiol-mediated neuroprotection. 1471 19

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivo through light activation of the insulin receptor beta-subunit. In this study, we have cloned the 41 kDa cytoplasmic region of the retinal insulin receptor (IRbeta) and used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to demonstrate the interaction between the p85 subunit of PI3K and the cytoplasmic region of IRbeta. Under conditions where IRbeta autophosphorylates, substitution of Y1322F and M1325P in IRbeta resulted in the abolition of p85 binding to the IRbeta, confirming that the p85 subunit of PI3K binds to Y1322. The binding site for p85 on IRbeta was also confirmed in the yeast three-hybrid system. Using the C-terminal region of IRbeta (amino acids 1293-1343 encompassing the YHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined that the IRbeta-pYTHM motif interacts with p85. We also used retinal organ cultures to demonstrate insulin activation of the insulin receptor and subsequent binding of p85, measured through GST pull-down assays with p85 fusion proteins. Further, the Y960F mutant insulin receptor, which does not bind IRS-1, is capable of bringing down PI3K activity from retina lysates. On the other hand, in response to insulin, IRS-2 is able to interact with the p85 subunit of PI3K in the retina. These results suggest that multiple signaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.
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PMID:Interaction of the retinal insulin receptor beta-subunit with the p85 subunit of phosphoinositide 3-kinase. 1513 38

The cellular functions and regulation of phosphatidylinositol (PtdIns) 5-phosphate (5-P), the newest addition to the family of phosphoinositides (PIs), are still elusive. Here we have examined a plausible role of PtdIns 5-P as a signaling intermediate in acute insulin action. A wortmannin-insensitive transient increase of PtdIns 5-P mass levels that peaked at 10 min, and declined 20-30 min after insulin stimulation, was observed in both Chinese hamster ovary (CHO)-T cells stably expressing the insulin receptor and 3T3-L1 adipocytes. Similarly to insulin, found to induce a rapid disassembly of Texas-Red phalloidin-labeled actin stress fibers in CHO-T cells, microinjected PtdIns 5-P, but not other PIs, decreased the number and length of F-actin stress fibers in this cell type to a magnitude seen in response to insulin. Likewise, increases of PtdIns 5-P by ectopic expression of the PtdIns 5-P-producing enzyme PIKfyve yielded a similar effect. As with insulin, the PtdIns 5-P-induced loss of actin stress fibers was independent of PI 3-kinase activation. Furthermore, sequestration of functional PtdIns 5-P, either by ectopic expression of 3xPHD domains that bind selectively PtdIns 5-P or by microinjecting the GST-3xPHD fusion peptide, abrogated insulin-induced F-actin stress fiber disassembly in CHO-T cells. In 3T3-L1 adipocytes, microinjected PtdIns 5-P, but not other PIs, partially mimicked insulin's effect of translocating enhanced green fluorescent protein-GLUT4 to the cell surface. Conversely, insulin-induced myc-GLUT4 vesicle dynamics was arrested in the presence of coexpressed enhanced green fluorescent protein-3xPHD. Involvement of PIKfyve membrane recruitment, but not activation, and/or a decrease in PtdIns 4,5-bisphosphate levels are likely to be among the mechanisms underlying the insulin-induced PtdIns 5-P increase. Together, these results identify PtdIns 5-P as a novel key intermediate for insulin signaling in F-actin remodeling and GLUT4 translocation.
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PMID:Role for a novel signaling intermediate, phosphatidylinositol 5-phosphate, in insulin-regulated F-actin stress fiber breakdown and GLUT4 translocation. 1528 92

IGF-1 receptor (IGF1R) is a transmembrane tyrosine kinase, which is indispensable for cellular growth and differentiation. Using a recombinant GST-tagged cytosolic fragment of IGF1R (GST-IGFK), we now show that oxidation by low doses (50 muM) of hydrogen peroxide markedly inhibits maximum phosphate incorporation in autophosphorylation and substrate phosphorylation assays. A similar inhibition was observed on the activity of intact IGF1R after treatment of T-47D cells. These results are in sharp contrast to the positive influence of hydrogen peroxide on the highly homologous insulin receptor kinase, which was assayed for comparison. This reciprocal influence of physiologically relevant doses of hydrogen peroxide may have important implications on signal transduction of the closely related receptors for insulin and IGF-1.
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PMID:Hydrogen peroxide inhibits activity of the IGF-1 receptor kinase. 1680 64

The SH2B family has three members (SH2B1, SH2B2, and SH2B3) that contain conserved dimerization (DD), pleckstrin homology, and SH2 domains. The DD domain mediates the formation of homo- and heterodimers between members of the SH2B family. The SH2 domain of SH2B1 (previously named SH2-B) or SH2B2 (previously named APS) binds to phosphorylated tyrosines in a variety of tyrosine kinases, including Janus kinase-2 (JAK2) and the insulin receptor, thereby promoting the activation of JAK2 or the insulin receptor, respectively. JAK2 binds to various members of the cytokine receptor family, including receptors for GH and leptin, to mediate cytokine responses. In mice, SH2B1 regulates energy and glucose homeostasis by enhancing leptin and insulin sensitivity. In this work, we identify SH2B2beta as a new isoform of SH2B2 (designated as SH2B2alpha) derived from the SH2B2 gene by alternative mRNA splicing. SH2B2beta has a DD and pleckstrin homology domain but lacks a SH2 domain. SH2B2beta bound to both SH2B1 and SH2B2alpha, as demonstrated by both the interaction of glutathione S-transferase-SH2B2beta fusion protein with SH2B1 or SH2B2alpha in vitro and coimmunoprecipitation of SH2B2beta with SH2B1 or SH2B2alpha in intact cells. SH2B2beta markedly attenuated the ability of SH2B1 to promote JAK2 activation and subsequent tyrosine phosphorylation of insulin receptor substrate-1 by JAK2. SH2B2beta also significantly inhibited SH2B1- or SH2B2alpha-promoted insulin signaling, including insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1. These data suggest that SH2B2beta is an endogenous inhibitor of SH2B1 and/or SH2B2alpha, negatively regulating insulin signaling and/or JAK2-mediated cellular responses.
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PMID:Identification of SH2B2beta as an inhibitor for SH2B1- and SH2B2alpha-promoted Janus kinase-2 activation and insulin signaling. 1720 55

It is well known that insulin receptor substrates (IRS) act as a mediator for signal transduction of insulin, insulin-like growth factors, and several cytokines. To identify proteins that interact with IRS and modulate IRS-mediated signals, we performed yeast two-hybrid screening with IRS-1 as bait. Out of 109 cDNA-positive clones identified from a human placental cDNA library, two clones encoded 53BP2, p53-binding protein 2 (53BP2S), a short form splicing variant of the apoptosis-stimulating protein of p53 that possesses Src homology region 3 domain, and ankyrin repeats domain, and had been reported to interact with p53, Bcl-2, and NF-kappaB. Interaction of 53BP2S with IRS-1 was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays in COS-7 cells and 3T3-L1 adipocytes. The Src homology region 3 domain and ankyrin repeats domain of 53BP2S were responsible for its interaction with IRS-1, whereas the phosphotyrosine binding domain and a central domain (amino acid residues 750-861) of IRS-1 were required for its interaction with 53BP2S. In CHO-C400 cells, expression of 53BP2S reduced insulin-stimulated IRS-1 tyrosine phosphorylation with a concomitant enhancement of IRS-2 tyrosine phosphorylation. In addition, the amount of the phosphatidylinositol 3-kinase regulatory p85 subunit associated with tyrosine-phosphorylated proteins, and activation of Akt was enhanced by 53BP2S expression. Although 53BP2S also enhanced Akt activation in 3T3-L1 adipocytes, insulin-induced glucose transporter 4 translocation was markedly inhibited in accordance with reduction of insulin-induced AS160 phosphorylation. Together these data demonstrate that 53BP2S interacts and modulates the insulin signals mediated by IRSs.
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PMID:53BP2S, interacting with insulin receptor substrates, modulates insulin signaling. 1796 23


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