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C R Kahn

Publications and source records attributed to C R Kahn.

At least 145 records · Page 8Linked to original sources
PubMed1994

Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.

3T3 Cells↗
PubMed1994

Regulation of insulin receptor, insulin receptor substrate-1 and phosphatidylinositol 3-kinase in 3T3-F442A adipocytes. Effects of differentiation, insulin, and dexamethasone.

Insulin rapidly stimulates tyrosine kinase activity of its receptor resulting in phosphorylation of its cytosolic substrate insulin receptor substrate 1 (IRS-1), which in turn associates with and activates the enzyme phosphatidylinositol 3-kinase (PI 3-kinase). In the present study we have examined these three initial steps in insulin action during the differentiation of 3T3-F442A adipocytes and after treatment with dexamethasone or insulin. The differentiation of 3T3-F442A cells was characterized by a 13-fold increase in insulin receptor protein, a 9-fold increase in IRS-1, and a 10- and 4.5-fold increase in their insulin-stimulated phosphorylation, respectively. The mRNA expression of these two proteins showed a similar 8-fold increase during differentiation. In addition there was a 3.5-fold increase in PI 3-kinase protein [85 kilodalton (kDa) subunit] and a 16-fold increase in IRS-1-associated PI 3-kinase activity between day 0 and day 8 of differentiation. Dexamethasone (1 microM) treatment of differentiated cells induced a further 48% (P < 0.05) increase in insulin receptor level, but the autophosphorylation of the receptor was decreased by 31 +/- 1% (P < 0.02). At the same time there was a decrease by 56 +/- 4% (P < 0.005) in IRS-1 protein and by 31 +/- 1% (P < 0.001) in IRS-1 phosphorylation. The expression of insulin receptor mRNA was unchanged, but the expression of IRS-1 mRNA was decreased by approximately 75% after dexamethasone. By contrast, dexamethasone induced a 69% increase in the level of PI 3-kinase as determined by immunoblotting. The combined effect of decreased IRS-1 phosphorylation and increased PI 3-kinase protein was a minimal change (15% decrease) in the association/activation between IRS-1 and PI 3-kinase. Chronic treatment with 100 nM insulin induced a time- and dose-dependent decrease in insulin receptor and IRS-1 protein levels reaching a nadir of 34 +/- 5% (P < 0.005) and 39 +/- 5% (P < 0.01) of control levels after 24 h, respectively. There was an even more marked decrease in the phosphorylation level of these proteins. Chronic insulin treatment also produced a 30% decrease in PI 3-kinase protein levels and a approximately 50% decrease in the association/activation between IRS-1/PI 3-kinase. The expression of insulin receptor and IRS-1 mRNA was unchanged during chronic insulin treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1994

The level of insulin receptor tyrosine kinase activity modulates the activities of phosphatidylinositol 3-kinase, microtubule-associated protein, and S6 kinases.

The role of insulin receptor tyrosine kinase activity in stimulation of intracellular enzymes linked to insulin action [phosphatidylinositol 3-kinase (PtdIns 3-kinase), microtubule-associated protein (MAP) kinase, and S6 kinases] was studied in Chinese hamster ovary cells which overexpress wild type human insulin receptors, receptors with reduced kinase activity due to substitution of Phe for Tyr1146 (single-Phe), Tyr1150,1151 (double-Phe), and Tyr1146,1150,1151 (triple-Phe), or kinase-inactive receptors with a substitution of Ala for Lys1018 in the ATP binding site (A1018). We have previously shown that receptor autophosphorylation and kinase activity of these mutants were reduced by approximately 50, 65, 85, and 100%, respectively. Glycogen and DNA synthesis parallel the level of receptor autophosphorylation and kinase activity; however, receptor serine and threonine phosphorylation was independent of receptor tyrosine kinase activity and receptor internalization was completely dependent on maximal receptor kinase activity. Overexpression of the wild type insulin receptor increased both maximal insulin receptor substrate-1-associated and total insulin-stimulated PtdIns 3-kinase activity, as well as S6 and MAP kinase activities 2.0- to 3.6-fold. In addition there was a leftward shift of the dose-response curves for PtdIns 3-kinase and S6 kinases by approximately 10-fold. Expression of the single- and double-Phe mutant receptors also enhanced maximal PtdIns 3-kinase activity, but had no effect on insulin sensitivity, whereas expression of either the triple-Phe or kinase-inactive receptors did not enhance insulin stimulation or increase insulin sensitivity as compared to the control cells. When comparing the mutant and wild type receptors, differences in insulin sensitivity were least for insulin-stimulated MAP kinase and greatest for S6 kinase; with the latter there was greater than a 1000-fold difference in insulin sensitivity when cells that overexpress wild type vs. kinase-inactive insulin receptors were compared. Thus, the level of insulin receptor tyrosine autophosphorylation and kinase activity regulate both maximal activation and insulin sensitivity of these intracellular kinases in the insulin action pathway which may lead to glycogen and/or DNA synthesis. The differential sensitivity of these enzymes to changes in receptor activation suggests that they may be differently coupled to the receptor kinase.

Amino Acid Sequence↗
PubMed1994

Insulin receptor substrate-1 (IRS-1) distribution in the rat central nervous system.

Insulin receptor substrate 1 (IRS-1) is the primary cytosolic substrate of the insulin and insulin-like growth factor-I (IGF-I) receptors. Following tyrosine phosphorylation IRS-1 binds to and activates specific proteins containing SH2 domains. Using biochemical and immunocytochemical techniques, we have mapped the distribution of IRS-1 in the CNS of the adult rat and compared it with that of insulin and IGF-I receptors and phosphatidylinositol 3-kinase (PI-3 kinase), a signaling molecule functionally related to IRS-1. Immunoprecipitation and Western blotting experiments demonstrate the presence of substantial amounts of IRS-1, insulin receptor, and PI-3 kinase in the brain. IRS-1 immunoreactivity is widely distributed in neurons from several areas of the brain and spinal cord. The cerebral cortex, the hippocampus, many hypothalamic and thalamic nuclei, the basal ganglia, the cerebellar cortex, the brainstem nuclei, and the lamina X of the spinal cord are particularly rich of immunopositive nerve cells. In these areas most of the neurons immunoreactive for IRS-1 are also stained by either anti-insulin receptor or anti-IGF-I receptor antibodies as well as PI-3 kinase antiserum. IRS-1 immunostaining was very weak or totally absent in neurons of the olfactory bulb, the supraoptic and paraventricular nuclei, the mesencephalic trigeminal nucleus, and the granule cell layer of the cerebellum, despite the fact that these areas were immunolabeled with antibodies against insulin or IGF-I receptors and/or PI-3 kinase. These results show that neurons in the adult rat CNS are endowed with some of the components of the early signaling pathway for growth factors of the insulin/IGF-I family, although IRS-1 has a distribution distinct from that of the two receptors.

Animals↗
PubMed1994

[Characterization of the IRS-1 (insulin receptor substrate-1) gene and its promoter].

IRS-1 (insulin receptor substrate-1) is a major substrate of the insulin receptor. Rat and human IRS-1 cDNAs, and human and mouse IRS-1 genes have been cloned so far. They show high homology in nucleic acids and amino acids levels, which indicate the high conservation of IRS-1 across the species. Interestingly, the entire coding region is contained in the 1st exon in the IRS-1 gene. The promoter of the mouse IRS-1 gene lacks TATA and CAAT boxes but contains 9 potential Spl binding sites, indicating that IRS-1 is a "housekeeping" gene. By deletion analysis, two positively and two negatively regulating fragments are identified in the promoter. In cultured adipocytes, insulin and dexamethasone down regulate IRS-1 expression by different mechanisms. Insulin down regulates at the post-translational level by shortening the protein half life, and dexamethasone down regulates at the post-transcriptional level mainly by shortening the mRNA half life.

Animals↗
PubMed1993

Rad: a member of the Ras family overexpressed in muscle of type II diabetic humans.

To identify the gene or genes associated with insulin resistance in Type II (non-insulin-dependent) diabetes mellitus, subtraction libraries were prepared from skeletal muscle of normal and diabetic humans and screened with subtracted probes. Only one clone out of 4000 was selectively overexpressed in Type II diabetic muscle as compared to muscle of non-diabetic or Type I diabetic individuals. This clone encoded a new 29-kilodalton member of the Ras-guanosine triphosphatase superfamily and was termed Rad (Ras associated with diabetes). Messenger ribonucleic acid of Rad was expressed primarily in skeletal and cardiac muscle and was increased an average of 8.6-fold in the muscle of Type II diabetics as compared to normal individuals.

Amino Acid Sequence↗
PubMed1993

Substitution of the erbB-2 oncoprotein transmembrane domain activates the insulin receptor and modulates the action of insulin and insulin-receptor substrate 1.

The mechanism through which insulin binding to the extracellular domain of the insulin receptor activates the intrinsic tyrosine kinase in the intracellular domain of the protein is unknown. For the c-neu/erbB-2 (c-erbB-2) protooncogene, a single point mutation within the transmembrane (TM) domain converting Val-664 to Glu (erbB-2V-->E) results in elevated levels of tyrosine kinase activity and cellular transformation. We report the construction of a chimeric insulin receptor in which the TM domain of the receptor has been substituted with that encoded by erbB-2V-->E. When expressed in Chinese hamster ovary cells this chimeric receptor displays maximal levels of autophosphorylation and kinase activity in the absence of insulin. This activity results in an increase in the level of insulin-receptor substrate 1 phosphorylation but a down-regulation in insulin-receptor substrate 1 protein and desensitization to insulin stimulation of glycogen synthesis. By contrast, basal levels of DNA synthesis are elevated to levels approximately 60% of those observed in serum-stimulated cells. Over-expression of chimeric insulin receptors containing the c-erbB-2 TM domain or a single point mutation in the insulin receptor TM domain of Val-938-->Asp, on the other hand, shows none of these alterations. Thus, the TM domain encoded by erbB-2V-->E contains structural features that can confer ligand-independent activation in a heterologous protein. Constitutive activation of the insulin receptor results in a relative increase in basal levels of DNA synthesis, but an apparent resistance to the metabolic effects of insulin.

Amino Acid Sequence↗
PubMed1993

Optimizing transmembrane domain helicity accelerates insulin receptor internalization and lateral mobility.

Transmembrane (TM) domains of integral membrane proteins are generally thought to be helical. However, a Gly-Pro sequence within the TM domain of the insulin receptor is predicted to act as a helix breaker. CD analyses of model TM peptides in a lipid-like environment show that substitution of Gly and Pro by Ala enhances helicity. On this basis, Gly933 and Pro934 within the TM domain of the intact human insulin receptor were mutated to Ala (G-->A, P-->A, GP-->AA) to assess effects of altered helicity on receptor functions. Mutated and wild-type receptors, expressed stably in cultured CHO cells at equivalent levels, were properly assembled, biosynthetically processed, and exhibited similar affinities for insulin. Receptor autophosphorylation and substrate kinase activity in intact cells and soluble receptor preparations were indistinguishable. In contrast, insulin-stimulated receptor internalization was accelerated 2-fold for the GP-->AA mutant, compared to a wild-type control or the G-->A and P-->A mutants. Insulin degradation, which occurs during receptor endocytosis and recycling, was similarly elevated in cells transfected with GP-->AA mutant receptors. Fluorescence photobleaching recovery measurements showed that the lateral mobility of GP-->AA mutant receptors was also increased 2- to 3-fold. These results suggest that lateral mobility directly influences rates of insulin-mediated receptor endocytosis and that rates of endocytosis and lateral mobility are retarded by a kinked TM domain in the wild-type receptor. Invariance of Gly-Pro within insulin receptor TM domain sequences suggests a physiologic advantage for submaximal rates of receptor internalization.

Amino Acid Sequence↗
PubMed1993

Insulin receptor substrate 1 mediates insulin and insulin-like growth factor I-stimulated maturation of Xenopus oocytes.

Insulin and insulin-like growth factor I (IGF-I) initiate cellular functions by activating their homologous tyrosine kinase receptors. In most mammalian cell types, this results in rapid tyrosine phosphorylation of a high-molecular-weight substrate termed insulin receptor substrate 1 (IRS-1). Previous studies suggest that IRS-1 may act as a "docking" protein that noncovalently associates with certain signal-transducing molecules containing src homology 2 domains; however, direct evidence for the role of IRS-1 in the final biological actions of these hormones is still lacking. We have developed a reconstitution system to study the role of IRS-1 in insulin and IGF-I signaling, taking advantage of the fact that Xenopus oocytes possess endogenous IGF-I receptors but have little or no IRS-1, as determined by immunoblotting with anti-IRS-1 and antiphosphotyrosine antibodies. After microinjection of IRS-1 protein produced in a baculovirus expression system, tyrosyl phosphorylation of injected IRS-1 is stimulated by both insulin and IGF-I in a concentration-dependent manner, with IGF-I more potent than insulin. Furthermore, after IRS-1 injection, both hormones induce a maturation response that correlates well with the amount of injected IRS-1. By contrast, overexpression of human insulin receptors in the Xenopus oocytes does not enhance either IRS-1 phosphorylation or oocyte maturation response upon insulin stimulation. These results demonstrate that IRS-1 serves a critical role in linking IGF-I and insulin to their final cellular responses.

Animals↗
PubMed1993

Insulin stimulates serine and tyrosine phosphorylation in the juxtamembrane region of the insulin receptor.

Insulin-stimulated autophosphorylation of the cytoplasmic juxtamembrane region of the human insulin receptor was examined by Tricine/SDS-PAGE. Various mutant receptor molecules were used to identify two tryptic phosphopeptides associated with the juxtamembrane region which accounts for 15% of the autophosphorylation of partially purified insulin receptor. These phosphopeptides were immunoprecipitated with an antipeptide antibody against the juxtamembrane sequence and were phosphorylated exclusively on tyrosine. Substitution of both Tyr960 and Tyr953 with alanine eliminated insulin-stimulated phosphorylation of the juxtamembrane region without affecting tyrosine autophosphorylation in the C terminus or regulatory regions. Monosubstitution of Tyr960 with phenylalanine or alanine reduced phosphorylation in the juxtamembrane region by more than 50%, and manual Edman degradation indicated that Tyr960 was phosphorylated in wild-type receptor. In vivo, phosphorylation of the juxtamembrane region accounts for one-third of the insulin receptor phosphorylation and contains both phosphoserine and phosphotyrosine. Deletion of Tyr960 and 11 adjacent amino acids eliminated insulin-stimulated phosphorylation of the juxtamembrane region. Substitution of Tyr960 reduced this phosphorylation by more than 50%. The insulin receptor also undergoes serine phosphorylation outside of the juxtamembrane region which depends on the presence of Tyr1151. Together with our previous studies, this report suggests that phosphorylation of Tyr960 may play an important role in signal transduction by the insulin receptor.

Amino Acid Sequence↗
PubMed1993

Insulin induces the phosphorylation of nucleolin. A possible mechanism of insulin-induced RNA efflux from nuclei.

Insulin induces the serine phosphorylation of the nucleolar protein nucleolin at subnanomolar concentrations in differentiated 3T3-442A cells. The stimulation is biphasic with phosphorylation reaching a maximum at 10 pM insulin and then declining to only 40% of basal levels at insulin concentrations of 1 microM. These changes are rapid, reaching half-maximal after 4 min and maximal after 15 min of incubation. The cell-permeable casein kinase II inhibitor 5,6-dichlorobenzimidazole-riboside prevents the insulin-stimulated phosphorylation of nucleolin suggesting that casein kinase II may mediate this effect of the hormone. Insulin-like growth factor 1 mimics the action of insulin on dephosphorylation of nucleolin at nanomolar concentrations suggesting that the latter effect may be mediated by insulin-like growth factor 1 receptors. Insulin treatment of 3T3-442A cells also results in a stimulation of RNA efflux from isolated, intact cell nuclei. The dose dependence of insulin-induced nucleolin phosphorylation and insulin-stimulated RNA efflux from intact cell nuclei are almost identical. Insulin induces an increase in the RNA efflux at subnanomolar concentrations in 3T3-442A adipocytes, while high (micromolar) concentrations of insulin inhibited the efflux of RNA. These data indicate that insulin regulates the phosphorylation/dephosphorylation of nucleolin, possibly via stimulation of casein kinase II, and this may play a role in regulation of the RNA efflux from nuclei.

3T3 Cells↗
PubMed1993

ATP induces a conformational change of the 90-kDa heat shock protein (hsp90).

The 90-kDa heat shock protein (hsp90) is a well conserved, abundant cytosolic protein believed to be a "chaperone" of most steroid receptors. We have recently demonstrated that hsp90 has an ATP-binding site and autophosphorylating activity (Csermely, P., and Kahn, C. R. (1991) J. Biol. Chem. 266, 4943-4950). Circular dichroism analysis of highly purified hsp90 from rat liver shows that ATP induces an increase of beta-pleated sheet content of hsp90. Vanadate, molybdate, and heat treatment at 56 degrees C induce a similar change in the circular dichroism spectrum. Fourier transformed infrared spectroscopy reveals an ATP-induced increase in the interchain interactions of the 90-kDa heat shock protein due to an increase in its beta-pleated sheet content. In further studies we found that ATP: 1) decreases the tryptophan fluorescence of hsp90 by 11.6 +/- 1.9%; 2) increases the hydrophobic character of the protein as determined by its distribution between an aqueous phase and phenyl-Sepharose; and 3) renders hsp90 less susceptible to tryptic digestion. Our results suggest that hsp90 undergoes an "open-->closed" conformational change after the addition of ATP, analogous in many respects to the similar changes of the DnaK protein, the immunoglobulin heavy chain binding protein (BiP/GRP78), and hsp70. The ATP-induced conformational change of hsp90 may be important in regulating its association with steroid receptors and other cellular proteins.

Adenosine Triphosphate↗
PubMed1993

Modulation of expression of insulin and IGF-I receptor by Epstein-Barr virus and its gene products LMP and EBNA-2 in lymphocyte cell lines.

The receptors for insulin and insulin-like growth factor I (IGF-I) are two closely related integral membrane glycoproteins involved in signalling of cell growth and metabolism. We have used the unique paradigm of pairs of Burkitt lymphoma cell lines (BLO2, BL30, BL41) with or without Epstein-Barr Virus (EBV) infection and cells transfected with EBV-related genes to examine effects of EBV on expression of these receptors at the gene and protein functional level. In BL30 and BL41 cells, EBV infection increased surface insulin binding and total receptor number by 2- and 18-fold, respectively. By contrast, EBV infection decreased total IGF-I receptors by 29 to 87% in all three cell lines. In general, there was a correlation between total receptor concentration and the level of insulin or IGF-I receptor mRNAs, although in one cell line insulin binding increased while receptor mRNA levels decreased slightly, suggesting posttranslational effects. BL41 cells transfected with a vector expressing the EBV latent membrane protein (LMP) exhibited a 2.6- to 3.2-fold increase in insulin receptor expression, whereas cells transfected with EBNA-2 (one of the EBV nuclear antigens) alone exhibited no effect. However, EBNA-2 appears to be required for the EBV effect on insulin receptor expression since cells infected with a mutant virus, P3JHRI, which lacks the EBNA-2 gene failed to show an increase in insulin receptor number. These data indicate that EBV infection of lymphocytes increases expression of insulin receptors while simultaneously decreasing expression of IGF-I receptors. The magnitude and sometimes even the direction of change, depends on host cell factors. A maximal increase in insulin receptors appears to require the coordinate action of several of the EBV proteins including LMP and EBNA-2.

Antigens, Viral↗
PubMed1993

Insulin induces rapid accumulation of insulin receptors and increases tyrosine kinase activity in the nucleus of cultured adipocytes.

To better understand the mechanism by which insulin exerts effects on events at the cell nucleus, we have studied insulin receptors and tyrosine kinase activity in nuclei isolated by sucrose density gradient centrifugation following insulin treatment of differentiated 3T3-F442A cells. Insulin stimulated nuclear accumulation of insulin receptors by approximately threefold at 5 min. The half-maximal effect was observed with 1-10 nM insulin. Following insulin treatment, phosphotyrosine content associated with the nuclear insulin receptor was also increased by twofold at 5 min with a similar insulin concentration dependency. These nuclear insulin receptors differ from the membrane-associated insulin receptors in that they were not efficiently solubilized with 1% Triton X-100. During the same period of time, insulin stimulated nuclear tyrosine kinase activity toward the exogenous substrate poly Glu4:Tyr1 tenfold in a time-dependent manner reaching a maximum at 30 min. The insulin receptor substrate protein 1 (IRS-1) could not be detected in the nucleus by immunoblotting. However, a nuclear protein with M(r) approximately 220 kDa was tyrosine phosphorylated, and insulin further stimulated this process threefold > 30 mins. Surface labeling was performed to determine if the nuclear insulin receptors would emerge from the plasma membrane fraction. Using 125I-BPA-insulin with intact cells, the intensity of nuclear insulin receptor labeling was negligible and not increased throughout 30 min incubation at 37 degrees C. In contrast, there was an increase in labeled receptors in the microsomal fraction following insulin treatment. Taken together, these results indicate that insulin rapidly increases nuclear insulin receptor appearance and activates nuclear tyrosine kinase activity. The insulin-induced accumulation of nuclear insulin receptors cannot be accounted for by internalization of surface membrane receptors. These effects of insulin may play an important role in action of the hormone at the nuclear level.

3T3 Cells↗
PubMed1993

Two steps of insulin receptor internalization depend on different domains of the beta-subunit.

The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin-coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.

Amino Acid Sequence↗