Biomedical subjects

C R Kahn

Publications and source records attributed to C R Kahn.

At least 163 records · Page 9Linked to original sources

PubMed1993

Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase.

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.

Amino Acid Sequence↗

PubMed1993

Molecular determinants of insulin action.

Insulin rapidly stimulates tyrosine phosphorylation of a 185-kDa protein in most cell types. This protein, insulin receptor substrate 1 (IRS-1), has been implicated as the first postreceptor step in insulin signal transmission based on studies with insulin receptor mutants. In cell culture and in vitro, phosphorylated IRS-1 associates with the lipid-metabolizing enzyme phosphatidylinositol 3-kinase (PI 3-kinase), resulting in activation of this enzyme. Thus, the insulin receptor, IRS-1 and PI-3 kinase represent three of the earliest steps in insulin action at the cellular level. We have recently demonstrated that insulin is capable of stimulating PI 3-kinase activity in liver and muscle in vivo in animals and that IRS-1 phosphorylation may play a significant role in the association/activation with PI 3-kinase in vivo.

Animals↗

PubMed1993

Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus.

Insulin stimulates tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), which in turn binds to and activates phosphatidylinositol 3-kinase (PI 3-kinase). In the present study, we have examined these processes in animal models of insulin-resistant and insulin-deficient diabetes mellitus. After in vivo insulin stimulation, there was a 60-80% decrease in IRS-1 phosphorylation in liver and muscle of the ob/ob mouse. There was no insulin stimulation of PI 3-kinase (85 kD subunit) association with IRS-1, and IRS-1-associated PI 3-kinase activity was reduced 90%. Insulin-stimulated total PI 3-kinase activity was also absent in both tissues of the ob/ob mouse. By contrast, in the streptozotocin diabetic rat, IRS-1 phosphorylation increased 50% in muscle, IRS-1-associated PI 3-kinase activity was increased two- to threefold in liver and muscle, and there was a 50% increase in the p85 associated with IRS-1 after insulin stimulation in muscle. In conclusion, (a) IRS-1-associated PI 3-kinase activity is differentially regulated in hyperinsulinemic and hypoinsulinemic diabetic states; (b) PI 3-kinase activation closely correlates with IRS-1 phosphorylation; and (c) reduced PI 3-kinase activity may play a role in the pathophysiology of insulin resistant diabetic states, such as that seen in the ob/ob mouse.

Animals↗

PubMed1993

Modulation of insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase in liver and muscle of dexamethasone-treated rats.

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 phosphatidylinositol 3-kinase (PI 3-kinase), thus activating the enzyme. Glucocorticoid treatment is known to produce insulin resistance, but the exact molecular mechanism is unknown. In the present study we have examined the levels and phosphorylation state of the insulin receptor and IRS-1, as well as the association/activation between IRS-1 and PI 3-kinase in the liver and muscle of rats treated with dexamethasone. After dexamethasone treatment (1 mg/kg per d for 5 d), there was no change in insulin receptor concentration in liver of rats as determined by immunoblotting with antibody to the COOH-terminus of the receptor. However, insulin stimulation of receptor autophosphorylation determined by immunoblotting with antiphosphotyrosine antibody was reduced by 46.7 +/- 9.1%. IRS-1 and PI 3-kinase protein levels increased in liver of dexamethasone-treated animals by 73 and 25%, respectively (P < 0.05). By contrast, IRS-1 phosphorylation was decreased by 31.3 +/- 10.9% (P < 0.05), and insulin stimulated PI 3-kinase activity in anti-IRS-1 immunoprecipitates was decreased by 79.5 +/- 11.2% (P < 0.02). In muscle, the changes were less dramatic, and often in opposite direction of those observed in liver. Thus, there was no significant change in insulin receptor level or phosphorylation after dexamethasone treatment. IRS-1 and PI 3-kinase levels were decreased to 38.6 and 65.6%, respectively (P < 0.01 and P < 0.05). IRS-1 phosphorylation showed no significant change in muscle, but insulin-stimulated IRS-1 associated PI 3-kinase was decreased by 41%. Thus, dexamethasone has differential effects on the proteins involved in the early steps in insulin action in liver and muscle. In both tissues, dexamethasone treatment results in a reduction in insulin-stimulated IRS-1-associated P I3-kinase, which may play a role in the pathogenesis of insulin resistance at the cellular level in these animals.

Amino Acid Sequence↗

PubMed1993

Human skeletal muscle insulin receptor substrate-1. Characterization of the cDNA, gene, and chromosomal localization.

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has > 50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5'-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36-37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000-180,000 M(r) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Human corneal epithelial primary cultures and cell lines with extended life span: in vitro model for ocular studies.

PURPOSE: To develop an in vitro model of human corneal epithelium that can be propagated in serum-free medium that is tissue specific, species specific, and continuously available. METHODS: Primary explant cultures from human cadaver donor corneas were generated and subsequently infected with Adeno 12-SV40 (Ad12-SV40) hybrid virus or transfected with plasmid RSV-T. RESULTS: Several lines of human corneal epithelial cells with extended life span were developed and characterized. Propagation of both primary cultures and lines with extended life span, upon collagen membranes at an air-liquid interface, promoted multilayering, more closely approximating the morphology observed in situ. CONCLUSIONS: In vitro models, using primary cultures of corneal epithelium and lines of corneal epithelial cells with extended life span, retain a variety of phenotypic characteristics and may be used as an adjunct to ocular toxicology studies and as a tool to investigate corneal epithelial cell biology.

Adenoviruses, Human↗

PubMed1992

Insulin stimulation of phosphatidylinositol 3-kinase activity and association with insulin receptor substrate 1 in liver and muscle of the intact rat.

Growth factors stimulate the enzyme phosphatidylinositol (PI) 3-kinase in cells in culture. Insulin rapidly stimulates tyrosine phosphorylation of its endogenous substrate, insulin receptor substrate 1 (IRS-1), and in vitro IRS-1 associates with PI 3-kinase, thus activating the enzyme. We have examined whether insulin is capable of stimulating the PI 3-kinase pathway in two physiological target tissues for the actions of insulin in vivo, liver and skeletal muscle. After intraportal injection of insulin into anesthetized rats, there was a 2-fold stimulation of total hepatic PI 3-kinase activity in liver and muscle extracts and a 10- to 20-fold increase in PI 3-kinase activity immunoprecipitated with anti-IRS-1 antibodies. Stimulation of PI 3-kinase was accompanied by an association between this enzyme and IRS-1 as detected by immunoprecipitation of liver and muscle extracts with anti-IRS-1 antibodies and Western blotting with antibodies to the 85-kDa subunit of PI 3-kinase. Immunoprecipitation with anti-p85 antibodies and phosphotyrosine immunoblotting revealed no tyrosine phosphorylation of PI 3-kinase, but demonstrated co-precipitation of tyrosine-phosphorylated IRS-1, as well as another phosphotyrosine protein of approximately 135-140 kDa. Thus, IRS-1 phosphorylation plays a significant role in the activation of PI 3-kinase in vivo by insulin.

Animals↗

PubMed1992

Expression and function of IRS-1 in insulin signal transmission.

IRS-1 is a major insulin receptor substrate which may play an important role in insulin signal transmission. The mRNA for IRS-1 in rat cells and tissues is about 9.5 kilobases (kb). Rat liver IRS-1 was stably expressed in Chinese hamster ovary (CHO) cells (CHO/IRS-1). Although its calculated molecular mass is 131 kDa, IRS-1 from quiescent cells migrated between 165 and 170 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IRS-1 was phosphorylated strongly on serine residues and weakly on threonine residues before insulin stimulation. Insulin immediately stimulated tyrosine phosphorylation of IRS-1, and after 10-30 min with insulin its apparent molecular mass increased to 175-180 kDa. Expression of the human insulin receptor and rat IRS-1 together in CHO/IR/IRS-1 cells increased the basal serine phosphorylation of IRS-1 and strongly increased tyrosine phosphorylation during insulin stimulation. Purified insulin receptors directly phosphorylated baculovirus-produced IRS-1 exclusively on tyrosine residues. By immunofluorescence, IRS-1 was absent from the nucleus, but otherwise distributed uniformly before and after insulin stimulation. Some IRS-1 associated with the insulin receptor during insulin stimulation. In addition, a phosphatidylinositol 3'-kinase associated with IRS-1 during insulin stimulation, and this association was more sensitive to insulin in CHO cells overexpressing the insulin receptor (CHO/IR cells), more responsive to insulin to CHO/IRS-1 cells, and both sensitive and responsive in CHO/IR/IRS-1 cells. Similarly, insulin-stimulated DNA synthesis was more sensitive to insulin in CHO/IR cells, and more responsive in CHO/IRS-1 cells; however, insulin-stimulated DNA synthesis was sensitive but poorly responsive to insulin in CHO/IR/IRS-1 cells. Together, these results suggest that IRS-1 is a direct physiologic substrate of the insulin receptor and may play an important role in insulin signal transmission.

Amino Acid Sequence↗

PubMed1992

Insulin differentially regulates protein phosphotyrosine phosphatase activity in rat hepatoma cells.

We have studied the effect of insulin stimulation on phosphotyrosine phosphatase (PTPase) activity in the well-differentiated rat hepatoma cell line Fao. PTPase activity was measured using a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation. Of the PTPase activity in Fao cells, 14% was in the cytosolic fraction, whereas 86% was in the particulate fraction; this latter fraction also had a 4-fold higher specific activity. Purification of the particulate fraction by lectin chromatography resulted in a 50% increase in specific activity, although this glycoprotein-rich fraction contained only 1.5% of the total activity. Both the cytosolic and particulate PTPase fractions were active toward the tyrosyl-phosphorylated insulin receptor in vitro. The activity of the particulate fraction but not the cytosolic fraction was inhibited by addition of a micromolar concentration of a phosphorylated peptide corresponding to residues 1142-1153 of the human insulin receptor sequence. By contrast, addition of the nonphosphorylated peptide even at millimolar concentration was without effect. Both PTPase fractions were inhibited by Zn+ at similar concentrations, whereas the cytosolic PTPase activity was 10-fold more sensitive to vanadate inhibition. Treatment of cells with 100 nM insulin increased PTPase activity in the particulate fraction by 40% and decreased activity in the cytosolic fraction by 35%. These effects occurred within 15 min and were half-maximal at 3-4 nM insulin. When assessed as total activity, the magnitude of the changes in PTPase activity in the particulate and cytosolic fractions could not be explained on the basis of a translocation of PTPases between the two pools.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Insulin induces the phosphorylation of DNA-binding nuclear proteins including lamins in 3T3-F442A.

Insulin binding to its plasma membrane receptor stimulates a cascade of protein kinases and phosphatases which ultimately affects multiple processes in the membrane, cytosol, and nucleus of the cell, including transcription of specific genes. To gain insight into the relationship between the kinase cascade and the mechanism of insulin-induced nuclear events, we have studied the effect of insulin on the phosphorylation of DNA-binding nuclear proteins in differentiated NIH-3T3-F442A adipocytes. Insulin induced the phosphorylation of seven DNA-binding proteins: pp34, pp40, pp48, pp62, pp64, pp66, and pp72. The half-maximal response was observed at 10-30 min and reached its maximum at 60 min. The insulin-induced phosphorylation of each of these proteins was dose-dependent with ED50S of 2-10 nM. The phosphorylation of pp62, pp64, and pp72 took place on serine residues. On the basis of immunoprecipitation and immunoblotting experiments with anti-lamin antibodies, we found that the insulin-induced DNA-binding phosphoproteins pp62, pp64, pp66, and possibly pp48 were related to lamins A and C. The ED50 for insulin-stimulated lamin phosphorylation was approximately 10 nM, and phosphorylation was half-maximal at 30 min. The insulin-dependent phosphorylation of lamins and other DNA-binding proteins (pp34, pp40, and pp72) may play a mediatory role in the long-term effects of insulin.

Adipose Tissue↗

PubMed1992

Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study.

Type 2 diabetes mellitus is characterised by resistance of peripheral tissues to insulin and a relative deficiency of insulin secretion. To find out which is the earliest or primary determinant of disease, we used a minimum model of glucose disposal and insulin secretion based on intravenous glucose tolerance tests to estimate insulin sensitivity (SI), glucose effectiveness (ie, insulin-independent glucose removal rate, SG), and first-phase and second-phase beta-cell responsiveness in normoglycaemic offspring of couples who both had type 2 diabetes. 155 subjects from 86 families were followed-up for 6-25 years. More than 10 years before the development of diabetes, subjects who developed the disease had lower values of both SI (mean 3.2 [SD 2.4] vs 8.1 [6.7] 10(-3) I min-1 pmol-1 insulin; p < 0.0001) and SG (1.6 [0.9] vs 2.3 [1.2] 10(-2) min-1, p < 0.0001) than did those who remained normoglycaemic). For the subjects with both SI and SG below the group median, the cumulative incidence of type 2 diabetes during the 25 years was 76% (95% confidence interval 54-99). By contrast, no subject with both SI and SG above the median developed the disease. Subjects with low SI/high SG or high SI/low SG had intermediate risks. Insulin secretion, especially first phase, tended to be increased rather than decreased in this prediabetic phase and was appropriate for the level of insulin resistance. The development of type 2 diabetes is preceded by and predicted by defects in both insulin-dependent and insulin-independent glucose uptake; the defects are detectable when the patients are normoglycaemic and in most cases more than a decade before diagnosis of disease.

Adolescent↗

PubMed1992

Insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function.

We have studied a series of insulin receptor molecules in which the 3 tyrosine residues which undergo autophosphorylation in the kinase domain of the beta-subunit (Tyr1158, Tyr1162, and Tyr1163) were replaced individually, in pairs, or all together with phenylalanine or serine by in vitro mutagenesis. A single-Phe replacement at each of these three positions reduced insulin-stimulated autophosphorylation of solubilized receptor by 45-60% of that observed with wild-type receptor. The double-Phe replacements showed a 60-70% reduction, and substitution of all 3 tyrosine residues with Phe or Ser reduced insulin-stimulated tyrosine autophosphorylation by greater than 80%. Phosphopeptide mapping each mutant revealed that all remaining tyrosine autophosphorylation sites were phosphorylated normally following insulin stimulation, and no new sites appeared. The single-Phe mutants showed insulin-stimulated kinase activity toward a synthetic peptide substrate of 50-75% when compared with wild-type receptor kinase activity. Insulin-stimulated kinase activity was further reduced in the double-Phe mutants and barely detectable in the triple-Phe mutants. In contrast to the wild-type receptor, all of the mutant receptor kinases showed a significant reduction in activation following in vitro insulin-stimulated autophosphorylation. When studied in intact Chinese hamster ovary cells, insulin-stimulated receptor autophosphorylation and tyrosine phosphorylation of the cellular substrate pp185 in the single-Phe and double-Phe mutants was progressively lower with increased tyrosine replacement and did not exceed the basal levels in the triple-Phe mutants. However, all the mutant receptors, including the triple-Phe mutant, retained the ability to undergo insulin-stimulated Ser and Thr phosphorylation. Thus, full activation of the insulin receptor tyrosine kinase is dependent on insulin-stimulated Tris phosphorylation of the kinase domain, and the level of autophosphorylation in the kinase domain provides a mechanism for modulating insulin receptor kinase activity following insulin stimulation. By contrast, insulin stimulation of receptor phosphorylation on Ser and Thr residues by cellular serine/threonine kinases can occur despite markedly reduced tyrosine autophosphorylation.

Amino Acid Sequence↗

PubMed1992

The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities. Analysis with insulin and anti-receptor antibodies.

The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp 185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Anti-receptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.

Animals↗

PubMed1992

Substitution of the insulin receptor transmembrane domain with the c-neu/erbB2 transmembrane domain constitutively activates the insulin receptor kinase in vitro.

To examine the role of the transmembrane domain (TM) of the insulin receptor in insulin-induced receptor kinase activation, we prepared four mutated insulin receptors: 1) a Val938----Asp substitution (IR/TMv----D), 2) insertion of a 3-amino acid repeat (Val938-Phe939-Leu940) (IR/TM+3), or the entire TM was replaced by the corresponding domain of either the 3) platelet-derived growth factor (PDGF) receptor (IR/TMPDGFR) or 4) c-neu/erbB2 proto-oncogene product (IR/TMc-neu). Each mutant receptor was stably expressed in Chinese hamster ovary cells, assessed by fluorescence-activated cell sorting, insulin binding, and biosynthetic labeling. All mutant receptors exhibited normal affinity for insulin. Pulse-chase experiments showed that each proreceptor was processed into alpha- and beta-subunits, although the rate of IR/TMV----D conversion was reduced approximately 3-fold. With IR/TMPDGFR, IR/TMV----D, and IR/TM+3 basal and insulin-stimulated levels of autophosphorylation and tyrosine kinase activation were normal, both in wheat germ agglutinin (WGA)-purified receptor preparations and intact cells. By contrast, following WGA purification or isolation of crude membranes, IR/TMc-neu was a constitutively active autokinase and substrate kinase in vitro. However, in intact cells insulin-stimulated autophosphorylation and kinase activity appeared normal. We conclude that although there is considerable latitude in acceptable structure, residues within the insulin receptor transmembrane domain can play a functional role in regulation of insulin receptor tyrosine kinase activity.

Amino Acid Sequence↗

PubMed1992

Cysteine 647 in the insulin receptor is required for normal covalent interaction between alpha- and beta-subunits and signal transduction.

We have investigated the structural and functional properties of two mutant insulin receptors in which Cys647 and Cys682,683,685 have been replaced with Ser (IRS647 and IRS682,683,685, respectively). Compared with the wild-type receptor (IRWT), both mutant receptors displayed altered sensitivities to dithiothreitol with respect to insulin binding and reduction of oligomeric forms. Subunit composition of the oligomeric forms of the receptors as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-labeled receptors indicated that Cys682,683,685 are required for normal heterotetrameric structure and that Cys647 plays a major role in the normal covalent association of the alpha- and beta-subunits. Under nonreducing conditions, the affinity-labeled IRS647 migrated, almost exclusively, as a 230-kDa species which appeared to represent an alpha 2 form of the receptor. Furthermore, Chinese hamster ovary cells expressing IRS647 did not exhibit basal or insulin-stimulated autophosphorylation, suggesting that Cys647 is also required for signal transduction.

Affinity Labels↗

PubMed1992

Approaches to the molecular cloning of protein-tyrosine phosphatases in insulin-sensitive tissues.

The intrinsic tyrosyl kinase activity of the insulin receptor is regulated by a balance between insulin-induced receptor autophosphorylation, which stimulates the receptor kinase, and enzymatic dephosphorylation of the receptor, which deactivates its kinase activity. The cellular protein-tyrosine phosphatase (PTPase) enzymes responsible for reversing the activated state of the insulin receptor have not been characterized. Our laboratory is interested in identifying and cloning the specific PTPase(s) that regulate the phosphorylation state of the insulin receptor. This chapter will summarize the design and results of our initial molecular cloning studies to identify specific PTPases in insulin-sensitive tissues that may have a potential physiological role in insulin action and clinical insulin resistance.

Amino Acid Sequence↗

PubMed1992

Insulin stimulation of phosphatidylinositol 3-kinase activity maps to insulin receptor regions required for endogenous substrate phosphorylation.

We have studied the phosphatidylinositol 3-kinase (PtdIns 3-kinase) in insulin-stimulated Chinese hamster ovary (CHO) cells expressing normal (CHO/IR) and mutant human insulin receptors. Insulin stimulation of CHO/IR cells results in an increase in PtdIns 3-kinase activity associated with anti-phosphotyrosine (alpha PY) immunoprecipitates, which has been previously shown to correlate with the in vivo production of PtdIns(3,4)P2, and PtdIns(3,4,5)P3 (Ruderman, N., Kapeller, R., White, M.F., and Cantley, L.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). Stimulation was maximal within 1 min and showed a dose response identical to that of insulin receptor autophosphorylation. The PtdIns 3-kinase also associated with the insulin receptor in an insulin-stimulated manner, as approximately 50% of the total alpha PY-precipitable activity could be specifically immunoprecipitated with anti-insulin receptor antibody. Mutant insulin receptors displayed variable ability to stimulate the PtdIns 3-kinase, but in all cases the presence of PtdIns 3-kinase in alpha PY immunoprecipitates correlated closely with the tyrosyl phosphorylation of the endogenous substrate pp185. In CHO cells expressing a kinase-deficient mutant (IRA1018), there was no observable insulin stimulation of PtdIns 3-kinase activity in alpha PY immunoprecipitates and no tyrosyl phosphorylation of pp185. Substitution of Tyr1146 in the insulin receptor regulatory region with phenylalanine partially impaired receptor autophosphorylation, pp185 phosphorylation, and insulin-stimulated increases in alpha PY-precipitable PtdIns 3-kinase activity. In contrast, a deletion mutant lacking 12 amino acids from the juxtamembrane region (IR delta 960) displayed normal in vivo autophosphorylation but failed to stimulate the PtdIns 3-kinase or phosphorylate pp185. Finally, a mutant receptor from which the C-terminal 43 amino acids had been deleted (IR delta CT) exhibited normal insulin-stimulated autophosphorylation, pp185 phosphorylation, and stimulation of the PtdIns 3-kinase activity in alpha PY immunoprecipitates. These data suggest that the PtdIns 3-kinase is itself a substrate of the insulin receptor kinase or associates preferentially with a substrate. A comparison of the biological activities of the mutant receptors with their activation of the PtdIns 3-kinase furthermore suggests that the PtdIns 3-kinase may be linked to insulin's ability to regulate DNA synthesis and cell growth.

1-Phosphatidylinositol 4-Kinase↗

PubMed1992

Quantitative dissociation between EGF effects on c-myc and c-fos gene expression, DNA synthesis, and epidermal growth factor receptor tyrosine kinase activity.

The exact relationship between EGF-stimulated tyrosine phosphorylation, induction of the cellular proto-oncogenes c-myc and c-fos, and DNA synthesis remains uncertain. Madin-Darby Canine Kidney (MDCK) cells possess EGF receptor sites with high binding capacity, and in contrast to A431 cells, respond to EGF by increasing DNA synthesis. Following EGF stimulation of intact MDCK cells, there was a rapid and marked increase in the autophosphorylation of the EGF receptor. This was associated with an increase in the tyrosine phosphorylation of a 120 kDa phosphoprotein believed to be an endogenous substrate of this receptor kinase. The ED50 for stimulation of phosphorylation of pp120 was approximately 0.05 nM versus 1.0 nM for receptor autophosphorylation, consistent with amplification of signalling at this step in EGF action. Stimulation of DNA synthesis occurred after 12 to 24 hours and revealed even further amplification with an ED50 of about 0.1 nM. Intermediate between these events was a time-dependent activation of c-fos and c-myc gene expression. However, the ED50 for these processes was approximately 10 nM, indicating a relatively lower sensitivity of EGF for stimulation of proto-oncogene expression. Tyrphostin (RG 50864), a compound reported to inhibit specifically the EGF receptor kinase, completely blocked EGF stimulation of proto-oncogene induction. Interestingly, under the same experimental conditions, EGF receptor autophosphorylation was decreased only 60%. These data, along with the dose-response studies, indicate that proto-oncogene induction requires near maximal stimulation of EGF receptor autophosphorylation. They also suggest that, in MDCK cells, the EGF dependent induction of the c-fos and c-myc genes is not strictly correlated to the extent of EGF receptor autophosphorylation or EGF-stimulated DNA synthesis, and that EGF stimulation of DNA synthesis likely involves additional rate-limiting intermediate steps.

Animals↗