Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells.
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ABSTRACT:
SUBMITTER: Koshio O
PROVIDER: S-EPMC1148820 | biostudies-other | 1988 Feb
REPOSITORIES: biostudies-other
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Project description:Concanavalin A (ConA) stimulated the phosphorylation of the beta-subunit of the insulin receptor and an Mr-185,000 protein on serine and tyrosine residues in intact H-35 rat hepatoma cells. This Mr-185,000 protein whose phosphorylation was stimulated by ConA was identical to pp185, a protein reported previously to be a putative endogenous substrate for the insulin receptor tyrosine kinase in rat hepatoma cells. In Chinese hamster ovary (CHO) cells transfected with cDNA of the human insulin receptor, tyrosine-phosphorylation of pp185 was strongly enhanced by ConA compared with the controls, suggesting that the induction of tyrosine-phosphorylation of pp185 was due to stimulation of the insulin receptor kinase by ConA. Moreover, monovalent ConA only slightly induced the tyrosine-phosphorylation of pp185, which was enhanced by the addition of anti-ConA IgG, suggesting that ConA stimulated the insulin receptor kinase mainly by the receptor cross-linking or aggregation in intact cells. These data suggest that the insulin-mimetic action of ConA is related to the autophosphorylation and activation of the insulin receptor tyrosine kinase, as well as the subsequent phosphorylation of pp185 in intact cells.
| S-EPMC1131367 | biostudies-other
Project description:We studied the phosphorylation of the beta subunit of the insulin receptor in intact freshly isolated rat hepatocytes, labelled with [32P]Pi. Insulin receptors partially purified by wheat-germ agglutinin chromatography were immunoprecipitated with either antibodies to insulin receptor or antibodies to phosphotyrosine. Receptors derived from cells incubated in the absence of insulin contained only phosphoserine. Addition of insulin to hepatocytes led to a dose-dependent increase in receptor beta-subunit phosphorylation, with half-maximal stimulation being observed at 2 nM-insulin. Incubation of cells with 100 nM-insulin showed that, within 1 min of exposure to the hormone, maximal receptor phosphorylation occurred, which was followed by a slight decrease and then a plateau. This insulin-induced stimulation of its receptor phosphorylation was largely accounted for by phosphorylation on tyrosine residues. Sequential immunoprecipitation of receptor with anti-phosphotyrosine antibodies and with anti-receptor antibodies, and phosphoamino acid analysis of the immunoprecipitated receptors, revealed that receptors that failed to undergo tyrosine phosphorylation were phosphorylated on serine residues. The demonstration of a functional hormone-sensitive insulin-receptor kinase in normal cells strongly supports a role for this receptor enzymic activity in mediating biological effects of insulin.
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Project description:Circadian rhythms in animals are regulated at the level of individual cells and by systemic signaling to coordinate the activities of multiple tissues. The circadian pacemakers have several physiological outputs, including daily locomotor rhythms. Several redox-active compounds have been found to function in regulation of circadian rhythms in cells, however, how particular compounds might be involved in regulating specific animal behaviors remains largely unknown. Here the effects of hydrogen peroxide on Drosophila movement were analyzed using a recently developed three-dimensional real-time multiple fly tracking assay. Both hydrogen peroxide feeding and direct injection of hydrogen peroxide caused increased adult fly locomotor activity. Continuous treatment with hydrogen peroxide also suppressed daily locomotor rhythms. Conditional over-expression of the hydrogen peroxide-producing enzyme superoxide dismutase (SOD) also increased fly activity and altered the patterns of locomotor activity across days and weeks. The real-time fly tracking system allowed for detailed analysis of the effects of these manipulations on behavior. For example, both hydrogen peroxide feeding and SOD over-expression increased all fly motion parameters, however, hydrogen peroxide feeding caused relatively more erratic movement, whereas SOD over-expression produced relatively faster-moving flies. Taken together, the data demonstrate that hydrogen peroxide has dramatic effects on fly movement and daily locomotor rhythms, and implicate hydrogen peroxide in the normal control of these processes.
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Project description:The function of protein products generated from intramembraneous cleavage by the ?-secretase complex is not well defined. The ?-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in A?, a transmembrane (TM) peptide. Another protein that undergoes very similar ?-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the ?-secretase cleavage site, we then investigated whether the p75(??) peptide, which is a product of both ?- and ?-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(??) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy.
| S-EPMC5633122 | biostudies-literature
Project description:Although various groups have studied the phosphorylation mechanism of the insulin receptor tyrosine kinase (IRK), an unanimous picture has not yet emerged. In this work, we performed a computational study to gain further insights. We first built a structural model of the reactant complex with the guide of several crystal structures and previous computational studies of the cyclic AMP-dependent protein kinase. We then optimized the structure by performing geometry optimization using a quantum mechanical model containing nearly 300 atoms. A reaction path was then traced between the reactant and the product by using a multiple coordinate-driven method. The calculations mapped out a sequence of structural changes depicting the conversion of the reactant to the product. Analysis of the structural changes revealed the formation of a dissociative transition state and the involvement of a proton transfer from the hydroxyl group of the tyrosyl residue of the peptide substrate to a conserved aspartate in the active site of the enzyme. The proton transfer began well before the transition state was reached and finished only shortly before the product was completely formed. In addition, the formation of a hydrogen bonding network among Arg1136, Asp1132, the gamma-phosphate of ATP, and the tyrosine residue of the substrate appeared to hold the latter two in a near-attack position for reaction. The model estimated a reaction barrier of 14 kcal/mol, semiquantitatively in accord with experiment.
| S-EPMC2785087 | biostudies-literature
Project description:Recent studies indicate that oxidative stress mediates salt-sensitive hypertension. To test the hypothesis that the renal epithelial sodium channel (ENaC) is a target of oxidative stress, patch clamp techniques were used to determine whether ENaC in A6 distal nephron cells is regulated by hydrogen peroxide (H(2)O(2)). In the cell-attached configuration, H(2)O(2) significantly increased ENaC open probability (P(o)) and single-channel current amplitude but not the unit conductance. High concentrations of exogenous H(2)O(2) are required to elevate intracellular H(2)O(2), probably because catalase, the enzyme that promotes the decomposition of H(2)O(2) to H(2)O and O(2), is highly expressed in A6 cells. The effect of H(2)O(2) on ENaC P(o) was enhanced by 3-aminotriazole, a catalase inhibitor, and abolished by overexpression of catalase, indicating that intracellular H(2)O(2) levels are critical to produce the effect. However, H(2)O(2) did not directly activate ENaC in inside-out patches. The effects of H(2)O(2) on ENaC P(o) and amiloride-sensitive Na(+) current were abolished by inhibition of phosphatidylinositide 3-kinase (PI3K). Confocal microscopy data showed that H(2)O(2) elevated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) in the apical membrane by stimulating PI3K. Because ENaC is stimulated by PI(3,4,5)P(3), these data suggest that H(2)O(2) stimulates ENaC via PI3K-mediated increases in apical PI(3,4,5)P(3).
| S-EPMC3173163 | biostudies-literature
Project description:H2O2 and vanadate are known insulinomimetic agents. Together they induce insulin's bioeffects with a potency which exceeds that seen with insulin, vanadate or H2O2 alone. We have previously shown that a combination of H2O2 and vanadate, when added to intact cells, rapidly stimulates protein tyrosine phosphorylation, owing to the inhibitory effects of these agents on intracellular protein tyrosine phosphatases (PTPases). Employing Western blotting with anti-phosphotyrosine antibodies, we have now identified in Chinese-hamster ovary (CHO) cells transfected with a wild-type insulin-receptor gene (CHO.T cells) several proteins (e.g. pp180, 125, 100, 60 and 52) whose phosphotyrosine content is rapidly increased upon treatment of the cells with a combination of insulin and 3 mM-H2O2. Tyrosine phosphorylation of these and additional proteins was further potentiated when 100 microM-sodium orthovanadate was added together with H2O2. The effects of insulin, insulin/H2O2, and H2O2/vanadate on tyrosine phosphorylation were markedly decreased in CHO cells transfected with an insulin-receptor gene where the twin tyrosines 1162 and 1163 were replaced with phenylalanine (CHO.YF-3 cells). Similarly, most of these proteins failed to undergo enhanced tyrosine phosphorylation in parental CHO cells incubated in the presence of insulin or the insulinomimetic agents. Our findings suggest that inhibition of PTPase activity by H2O2/vanadate augments the autophosphorylation of tyrosines 1162 and 1163 of the insulin receptor kinase, leading to its activation in an insulin-independent manner. As a result, tyrosine phosphorylation of potential targets for this enzyme takes place. Failure of H2O2/vanadate to induce phosphorylation of these proteins in receptor mutants lacking these twin tyrosine residues supports this hypothesis.
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Project description:Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.
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Project description:To explain the insulin resistance induced by catecholamines, we studied the tyrosine kinase activity of insulin receptors in a state characterized by elevated noradrenaline concentrations in vivo, i.e. cold-acclimation. Insulin receptors were partially purified from brown adipose tissue of 3-week- or 48 h-cold-acclimated mice. Insulin-stimulated receptor autophosphorylation and tyrosine kinase activity of insulin receptors prepared from cold-acclimated mice were decreased. Since the effect of noradrenaline is mediated by cyclic AMP and cyclic AMP-dependent protein kinase, we tested the effect of the purified catalytic subunit of this enzyme on insulin receptors purified by wheat-germ agglutinin chromatography. The catalytic subunit had no effect on basal phosphorylation, but completely inhibited the insulin-stimulated receptor phosphorylation. Similarly, receptor kinase activity towards exogenous substrates such as histone or a tyrosine-containing copolymer was abolished. This inhibitory effect was observed with receptors prepared from brown adipose tissue, isolated hepatocytes and skeletal muscle. The same results were obtained on epidermal-growth-factor receptors. Further, the catalytic subunit exerted a comparable effect on the phosphorylation of highly purified insulin receptors. To explain this inhibition, we were able to rule out the following phenomena: a change in insulin binding, a change in the Km of the enzyme for ATP, activation of a phosphatase activity present in the insulin-receptor preparation, depletion of ATP, and phosphorylation of a serine residue of the receptor. These results suggest that the alteration in the insulin-receptor tyrosine kinase activity induced by cyclic AMP-dependent protein kinase could contribute to the insulin resistance produced by catecholamines.
| S-EPMC1148077 | biostudies-other
Project description: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.
| S-EPMC1223928 | biostudies-other