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Growth inhibition signalled through the interleukin-4/interleukin-13 receptor complex is associated with tyrosine phosphorylation of insulin receptor substrate-1.

ABSTRACT: Induction of growth inhibition in human colorectal carcinoma cell lines by interleukin (IL)-4 and IL-13 was associated with the neophosphorylation of a 170 kDa cellular protein, identified as insulin receptor substrate-1 (IRS-1) by immunoprecipitation. Tyrosine phosphorylation of IRS-I was also induced by insulin and insulin-like growth factor I. Sublines of colorectal carcinoma cells unresponsive to growth modulation by IL-4, IL-13 or insulin-like growth factor I-induced growth did not phosphorylate IRS-1. A functional, multimeric IL-4 receptor complex was present on all carcinoma cell lines with a subunit composition of 65 kDa, 75 kDa and the previously characterized 130 kDa band as demonstrated by affinity cross-link with 126I labelled IL-4. The 65 kDa subunit is novel whereas the 75 kDa band represents the common IL-2 receptor gama-chain the novel 65 kDa receptor was present as a double band and bound primarily 125I-labelled IL-13. The present study demonstrates the involvement of a novel chain other than the gama-chain in the receptor complexes of IL-4 and IL-13 and and post-receptor tyrosine phosphorylation of IRS-1. The association of IRS-1 with growth inhibitory signals in carcinoma cells suggests a novel mechanism of tumour growth control.

SUBMITTER: Schnyder B

PROVIDER: S-EPMC1217273 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

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

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.

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Ethanol inhibits insulin receptor substrate-1 tyrosine phosphorylation and insulin-stimulated neuronal thread protein gene expression.

Project description:Neuronal thread proteins (NTPs) are molecules that accumulate in the brains of patients with Alzheimer's disease, and may play a key role in both normal and neurodegenerative neuritic sprouting. In this investigation we determined whether NTP expression is up-regulated by insulin, an important neurotrophic factor that stimulates differentiation-associated neurite outgrowth, and studied the effects of ethanol, a known inhibitor of growth factor receptor tyrosine phosphorylation, on NTP expression and insulin-mediated signal transduction cascade in neuronal [primitive neuroectodermal tumour cell line 2; (PNET2)] cells. PNET2 cells were treated with 50 m-units/ml insulin in the presence or absence of 100 mM ethanol for 0.2-96 h, and cell proliferation and expression of NTP molecules were investigated by metabolic labelling, immunoprecipitation and immunohistochemical staining. Insulin stimulation resulted in an immediate increase in the levels of three (38, 18 and 15 kDa) of five NTP species (the others were of 26 and 21 kDa), followed by a decline in expression within 120 min; however, studies performed up to 96 h of culture demonstrated up-regulation by insulin of all five NTP species. Ethanol either abolished or severely muted the short- and long-term insulin-mediated upregulation of NTP expression, and substantially reduced insulin-mediated neuronal differentiation. The effects of ethanol on NTP gene expression were associated with impaired insulin-mediated tyrosine phosphorylation of both the insulin receptor beta subunit and the insulin receptor substrate-1 (IRS-1), resulting in decreased association of phosphatidylinositol 3-kinase with IRS-1. The findings suggest that ethanol may inhibit NTP expression associated with central nervous system neuronal differentiation by uncoupling the IRS-1-mediated insulin signal transduction pathway.

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Insulin receptor substrate-2 deficiency impairs brain growth and promotes tau phosphorylation.

Project description:Insulin resistance and diabetes might promote neurodegenerative disease, but a molecular link between these disorders is unknown. Many factors are responsible for brain growth, patterning, and survival, including the insulin-insulin-like growth factor (IGF)-signaling cascades that are mediated by tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. Irs2 signaling mediates peripheral insulin action and pancreatic beta-cell function, and its failure causes diabetes in mice. In this study, we reveal two important roles for Irs2 signaling in the mouse brain. First, disruption of the Irs2 gene reduced neuronal proliferation during development by 50%, which dissociated brain growth from Irs1-dependent body growth. Second, neurofibrillary tangles containing phosphorylated tau accumulated in the hippocampus of old Irs2 knock-out mice, suggesting that Irs2 signaling is neuroprotective. Thus, dysregulation of the Irs2 branch of the insulin-Igf-signaling cascade reveals a molecular link between diabetes and neurodegenerative disease.

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A computational study of the phosphorylation mechanism of the insulin receptor tyrosine kinase.

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.

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Anti-(insulin receptor) monoclonal antibody-stimulated tyrosine phosphorylation in cells transfected with human insulin receptor cDNA.

Project description:The effects of insulin and anti-(insulin receptor) monoclonal antibodies on tyrosine phosphorylation were investigated in fibroblasts transfected with human insulin receptor cDNA (NIH 3T3HIR3.5 cells) using anti-phosphotyrosine immunoblotting. Insulin increased levels of tyrosine phosphorylation in two major proteins of molecular mass 97 kDa (pp97, assumed to be the insulin receptor beta-subunit) and 185 kDa (pp185). Insulin-mimetic anti-receptor antibodies also stimulated tyrosine phosphorylation of both pp97 and pp185. The observation of antibody-stimulated pp97 phosphorylation, as detected by immunoblotting, is in contrast with previous data which failed to show receptor autophosphorylation in NIH 3T3HIR3.5 cells labelled with [32P]P1. The effect of insulin on pp97 was maximal within 1 min, but the response to antibody was apparent only after a lag of 1-2 min and rose steadily over 20 min. The absolute level of antibody-stimulated phosphorylation of both pp97 and pp185 after 20 min was only about 20% of the maximum level induced by equivalent concentrations of insulin, even at concentrations of antibody sufficient for full occupancy of receptors. Another insulin-mimetic agent, wheat-germ agglutinin, stimulated receptor autophosphorylation with kinetics similar to those produced by the antibody. It is suggested that the relatively slow responses to both agents may be a function of the dependence on receptor cross-linking. These data are consistent with a role for the insulin receptor tyrosine kinase activity in the mechanism of action of insulin-mimetic anti-receptor antibodies.

| S-EPMC1131483 | biostudies-other

Tyrosine phosphorylation of mig6 reduces its inhibition of the epidermal growth factor receptor.

Project description:Under physiological conditions, epidermal growth factor receptor (EGFR) tyrosine kinase activity is tightly controlled through the coordinated action of both positive and negative regulators. Aberrant EGFR activation occurs frequently in many cancer types, and the endogenous EGFR feedback inhibitor, Mig6/RALT, is more efficiently phosphorylated by oncogenic EGFR variants. We have utilized expressed protein ligation to generate semisynthetic Tyr394 phosphorylated and unphosphorylated forms of the Mig6 protein and shown that phosphorylation of Mig6 reduces its ability to inhibit purified, near full-length EGFR (tEGFR). We also demonstrate that the kinetic parameters of tEGFR are similar whether solubilized in detergent or reconstitutued in nanodisc bilayers. These findings suggest a mechanism by which EGFR and its family members evade negative regulation by Mig6 under pathological conditions.

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