Monoclonal antibodies mimic insulin activation of ribosomal protein S6 kinase without activation of insulin receptor tyrosine kinase. Studies in cells transfected with normal and mutant human insulin receptors

The effects of species-specific monoclonal antibodies to the human insulin receptor on ribosomal protein S6 phosphorylation were studied in rodent cell lines transfected with human insulin receptors. First, Swiss mouse 3T3 fibroblasts expressing normal human insulin receptors (3T3/HIR cells) were studied. Three monoclonal antibodies, MA-5, MA-20, and MA-51, activated S6 kinase in these cells but had no effects in untransfected 3T3 cells. Both insulin and MA-5, the most potent antibody, activated S6 kinase in a similar time- and dose-dependent manner. To measure S6 phosphorylation in vivo, 3T3/HIR cells were preincubated with [32P]Pi and treated with insulin and MA-5. Both agents increased S6 phosphorylation, and their tryptic phosphopeptide maps were similar. MA-5 and the other monoclonal antibodies, unlike insulin, failed to stimulate insulin receptor tyrosine kinase activity either in vitro or in vivo. Moreover, unlike insulin, they failed to increase the tyrosine phosphorylation of the endogenous cytoplasmic protein, pp 185. Next, HTC rat hepatoma cells, expressing a human insulin receptor mutant that had three key tyrosine autophosphorylation sites in the beta-subunit changed to phenylalanines (HTC-IR-F3 cells), were studied. In this cell line but not in untransfected HTC cells, monoclonal antibodies activated S6 kinase without stimulating either insulin receptor autophosphorylation or the tyrosine phosphorylation of pp 185. These data indicate, therefore, that monoclonal antibodies can activate S6 kinase and then increase S6 phosphorylation. Moreover, they suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may not be crucial for activation of S6 kinase by the insulin receptor.     

Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro

Phosphorylation of the insulin receptor was studied in intact well differentiated hepatoma cells (Fao) and in a solubilized and partially purified receptor preparation obtained from these cells by affinity chromatography on wheat germ agglutinin agarose. Tryptic peptides containing the phosphorylation sites of the beta-subunit of the insulin receptor were analyzed by reverse-phase high performance liquid chromatography. Phosphoamino acid content of these peptides was determined by acid hydrolysis and high voltage electrophoresis. Separation of the phosphopeptides from unstimulated Fao cells revealed one major and two minor phosphoserine-containing peptides and a single minor phosphothreonine-containing peptide. Insulin (10(-7) M) increased the phosphorylation of the beta-subunit of the insulin receptor 3- to 4-fold in the intact Fao cell. After insulin stimulation, two phosphotyrosine-containing peptides were identified. Tyrosine phosphorylation reached a steady state within 20 s after the addition of insulin and remained nearly constant for 1 h. Under our experimental conditions, no significant change in the amount of [32P]phosphoserine or [32P]phosphothreonine associated with the beta-subunit was found during the initial response of cells to insulin. When the insulin receptor was extracted from the Fao cells and incubated in vitro with [gamma-32P]ATP and Mn2+, very little phosphorylation occurred in the absence of insulin. In this preparation, insulin rapidly stimulated autophosphorylation of the receptor on tyrosine residues only and high performance liquid chromatography analysis of the beta-subunit digested with trypsin revealed one minor and two major phosphopeptides. The elution position of the minor peptide corresponded to that of the major phosphotyrosine-containing peptide obtained from the beta-subunit of the insulin-stimulated receptor labeled in vivo. In contrast, the elution position of one of the major phosphopeptides that occurred during in vitro phosphorylation corresponded to the minor phosphotyrosine-containing peptide phosphorylated in vivo. The other major in vitro phosphotyrosine-containing peptide was not detected in vivo. Our results indicate that: tyrosine phosphorylation of the insulin receptor occurs rapidly following insulin binding to intact cells; the level of tyrosine phosphorylation remains constant for up to 1 h; the specificity of the receptor kinase or accessibility of the phosphorylation sites are different in vivo and in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

The insulin receptor functions normally in Chinese hamster ovary cells after truncation of the C terminus.

We studied the structure and function of the human insulin receptor (IR) and a mutant which lacked the last 43 amino acids of the beta-subunit (IR delta ct). This deletion removed tyrosine (Tyr1322, Tyr1316) and threonine (Thr1336) phosphorylation sites. In Chinese hamster ovary (CHO) cells, insulin binding to the mutant receptor was normal, and [35S]methionine labeling indicated that both the IR and IR delta ct were processed normally; however, the beta-subunit of IR delta ct was 5 kDa smaller than that of the IR. The time course of insulin-stimulated autophosphorylation of the partially purified IR delta ct was normal, but the maximum autophosphorylation was reduced 20-30%. Tryptic phosphopeptide mapping confirmed the absence of the C-terminal phosphorylation sites and indicated that phosphorylation of the regulatory region (Tyr1146, Tyr1150, Tyr1151) occurred normally; kinase activity of the IR and IR delta ct was activated normally by insulin-stimulated autophosphorylation. In the intact CHO cells, insulin-stimulated serine and threonine phosphorylation of the IR delta ct was reduced 20%, suggesting that most Ser/Thr phosphorylation sites are located outside of the C terminus. During insulin stimulation, the wild-type and mutant insulin receptor activated the phosphatidylinositol 3-kinase. Moreover, insulin itself or human-specific anti-insulin receptor antibodies stimulated glycogen and DNA synthesis equally in both CHO/IR and CHO/IR delta ct cells. These data suggest that the C terminus plays a minimal role in IR function and signal transmission in CHO cells.     

Alteration of intramolecular disulfides in insulin receptor/kinase by insulin and dithiothreitol: insulin potentiates the apparent dithiothreitol-dependent subunit reduction of insulin receptor

Dithiothreitol (DTT) was observed to increase both beta-subunit autophosphorylation and exogenous substrate phosphorylation of the insulin receptor in the absence of insulin. The natural protein reducing agent thioredoxin was also observed to increase the insulin receptor beta-subunit autophosphorylation. The activation of the insulin receptor/kinase by both DTT and thioredoxin was found to be additive with that of insulin. Further, the increase in the insulin receptor beta-subunit autophosphorylation in the presence of DTT and insulin was demonstrated to be due to an increase in the initial rate of autophosphorylation without alteration in the extent of phosphorylation. Similarly, the increase in the exogenous substrate phosphorylation was due to an increase in the Vmax of phosphorylation without significant effect on the apparent Km of substrate binding. In the presence of relatively low concentrations of DTT, insulin was found to potentiate the apparent insulin receptor subunit reduction of the native alpha 2 beta 2 heterotetrameric complex into alpha beta heterodimers, when observed by silver staining of sodium dodecyl sulfate-polyacrylamide gels. N-[3H]Ethylmaleimide ([3H]NEM) labeling in the absence of DTT pretreatment demonstrated that only the beta subunit had accessible sulfhydryl group(s). However, treatment of insulin receptors with DTT increased the amount of [3H]NEM labeling in the beta subunit as well as exposing sites on the alpha subunit. Further, incubation of the insulin receptors with the combination of DTT and insulin also demonstrated the apparent insulin-potentiated subunit reduction without any increase in the total amount of [3H]NEM labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Insulin-mimetic effect of trypsin on the insulin receptor tyrosine kinase in intact adipocytes

It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.     

Regulation of biological functions by an insulin receptor monoclonal antibody in insulin receptor beta-subunit mutants.

We investigated the effects of MA-5, a human-specific monoclonal antibody to the insulin receptor alpha-subunit, on transmembrane signaling in cell lines transfected with and expressing both normal human insulin receptors and receptors mutated in their beta-subunit tyrosine kinase domains. In cell lines expressing normal human insulin receptors, MA-5 stimulated three biological functions: aminoisobutyric acid (AIB) uptake, thymidine incorporation, and S6 kinase activation. Under conditions where these biological functions were stimulated, there was no detectable stimulation of receptor tyrosine kinase. We then combined the use of this monoclonal antibody with cells expressing insulin receptors with mutations in the beta-subunit tyrosine kinase domain; two of ATP binding site mutants V1008 (Gly----Val) and M1030 (Lys----Met) and one triple-tyrosine autophosphorylation site mutant F3 (Tyr----Phe at 1158, 1162, and 1163). In cells expressing V1008 receptors, none of the three biological functions of insulin was stimulated. In cells expressing M1030 receptors, AIB uptake was stimulated to a small, but significant, extent whereas the other two functions were not. In cells expressing F3 receptors, AIB uptake and S6 kinase activation, but not thymidine incorporation, were fully stimulated. The data suggest, therefore, that (1) activation of insulin receptor tyrosine kinase may not be a prerequisite for signaling of all the actions of insulin and (2) there may be multiple signal transduction pathways to account for the biological actions of insulin.     

A cascade of tyrosine autophosphorylation in the beta-subunit activates the phosphotransferase of the insulin receptor

We identified the major autophosphorylation sites in the insulin receptor and correlated their phosphorylation with the phosphotransferase activity of the receptor on synthetic peptides. The receptor, purified from Fao hepatoma cells on immobilized wheat germ agglutinin, undergoes autophosphorylation at several tyrosine residues in its beta-subunit; however, anti-phosphotyrosine antibody (alpha-PY) inhibited most of the phosphorylation by trapping the initial sites in an inactive complex. Exhaustive trypsin digestion of the inhibited beta-subunit yielded two peptides derived from the Tyr-1150 domain (Ullrich, A, Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) called pY4 and pY5. Both peptides contained 2 phosphotyrosyl residues (2Tyr(P], one corresponding to Tyr-1146 and the other to Tyr-1150 or Tyr-1151. In the absence of the alpha-PY additional sites were phosphorylated. The C-terminal domain of the beta-subunit contained phosphotyrosine at Tyr-1316 and Tyr-1322. Removal of the C-terminal domain by mild trypsinolysis did not affect the phosphotransferase activity of the beta-subunit suggesting that these sites did not play a regulatory role. Full activation of the insulin receptor during in vitro assay correlated with the appearance of two phosphopeptides in the tryptic digest of the beta-subunit, pY1 and pY1a, that were inhibited by the alpha-PY. Structural analysis suggested that pY1 and pY1a were derived from the Tyr-1150 domain and contained 3 phosphotyrosyl residues (3Tyr(P] corresponding to Tyr-1146, Tyr-1150, and Tyr-1151. The phosphotransferase of the receptor that was phosphorylated in the presence of alpha-PY at 2 tyrosyl residues in the Tyr-1150 domain was not fully activated during kinase assays carried out with saturating substrate concentrations which inhibited further autophosphorylation. During insulin stimulation of the intact cell, the 3Tyr(P) form of the Tyr-1150 domain was barely detected, whereas the 2Tyr(P) form predominated. We conclude that 1) autophosphorylation of the insulin receptor begins by phosphorylation of Tyr-1146 and either Tyr-1150 or Tyr-1151; 2) progression of the cascade to phosphorylation of the third tyrosyl residue fully activates the phosphotransferase during in vitro assay; 3) in vivo, the 2Tyr(P) form predominates, suggesting that progression of the autophosphorylation cascade to the 3Tyr(P) form is regulated during insulin stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two-dimensional phosphopeptide analysis of the autophosphorylation cascade of a soluble insulin receptor tyrosine kinase. The tyrosines phosphorylated are typical of those observed following phosphorylation of the heterotetrameric insulin receptor in intact cells.

A soluble derivative of the human insulin receptor cytoplasmic domain, as expressed in insect cells via a Baculovirus vector, is an active protein-tyrosine kinase. In the present study, we find that three forms of the enzyme (48, 43, and 38 kDa) can be partially purified by MonoQ fast protein liquid chromatography. Two-dimensional thin layer phosphopeptide mapping reveals that the 48-kDa enzyme undergoes a rapid autophosphorylation on the same tyrosines (residues 1158, 1162, 1163, 1328, and 1334) that have previously been shown to be major autophosphorylation sites on the native insulin receptor beta-subunit in intact cells. Furthermore, the 48- and 43-kDa proteins are phosphorylated on serine residues by a serine kinase(s) that copurifies through MonoQ fast protein liquid chromatography. Tyrosine autophosphorylation sites 1328 and 1334 and virtually all serine phosphorylation sites are absent in the 38-kDa kinase. Partial tryptic proteolysis of the 48-kDa kinase generates a core 38-kDa enzyme that undergoes autophosphorylation almost exclusively on tyrosines 1158, 1162, and 1163. Phosphorylation of these tyrosine residues occurs in a cascade manner analogous to that found in the intact insulin receptor beta-subunit.     

Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.

Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.     

Incorporation of the purified human placental insulin receptor into phospholipid vesicles

Purified human placental insulin receptors were incorporated into small unilamellar phospholipid vesicles by the addition of n-octyl beta-glucopyranoside solubilized phospholipids, followed by removal of the detergent on a Sephadex G-50 gel filtration column and extensive dialysis. The vesicles have an average diameter of 142 +/- 24 nm by Sephacryl S-1000 gel filtration chromatography and 119 +/- 20 nm by transmission electron microscopy. These vesicles are impermeant to small molecules as indicated by their ability to retain [gamma-32P]ATP, which could be released by the addition of 0.05% Triton X-100. Detergent permeabilization or freeze-thawing of the insulin receptor containing vesicles in the presence of 125I-insulin indicated that approximately 75% of the insulin binding sites were oriented right side out (extravesicularly). Sucrose gradient centrifugation of insulin receptors incorporated at various protein to phospholipid mole ratios demonstrated that the insulin receptors were inserted into the phospholipid bilayer structure in a concentration-dependent manner. Addition of [gamma-32P]ATP to the insulin receptor containing vesicles was relatively ineffective in promoting the autophosphorylation of the beta subunit in the absence or presence of insulin. Permeabilization of the vesicles with low detergent concentrations, however, stimulated the beta-subunit autophosphorylation approximately 2-fold in the absence and 10-fold in the presence of insulin. Insulin-stimulated beta-subunit autophosphorylation was also observed under conditions such that 94% of those vesicles containing insulin receptors had a single receptor per vesicle, suggesting that the initial beta-subunit autophosphorylating activity is intramolecular. Phospho amino acid analysis of the vesicle-incorporated insulin receptors demonstrated that the basal and insulin-stimulated beta-subunit autophosphorylation occurs exclusively on tyrosine residues. It is concluded that when purified insulin receptors are incorporated into a phospholipid bilayer, they insert into the vesicles primarily in the same orientation as occurs in the plasma membrane of intact cells and retain insulin binding as well as insulin-stimulated beta-subunit autophosphorylating activities.     

Biologic activities of naturally occurring human insulin receptor mutations. Evidence that metabolic effects of insulin can be mediated by a kinase-deficient insulin receptor mutant.

We have studied insulin receptor-mediated signaling in Chinese hamster ovary (CHO) cell transfectants that expressed either of two naturally occurring mutant human insulin receptors: Trp1200----Ser1200 and Ala1134----Thr1134. Compared with overexpressed normal human insulin receptors, both mutant receptors displayed normal processing and normal binding affinity; however, neither was capable of detectable insulin-stimulated autophosphorylation or tyrosine kinase activity toward endogenous (pp185) or exogenous substrates. Several biologic actions of insulin were evaluated in transfected cells. Compared with neomycin-only transfected CHO cells (CHO-NEO), cells expressing normal receptors demonstrated increased insulin sensitivity for 2-deoxyglucose uptake, [14C]glucose incorporation into glycogen, [3H]thymidine incorporation into DNA, and specific gene expression (accumulation of glucose transporter GLUT-1 mRNA). Cells expressing either Ser1200 or Thr1134 receptors showed no increase in insulin-stimulated thymidine incorporation or GLUT-1 mRNA accumulation compared with CHO-NEO. Surprisingly, cells expressing Ser1200 receptors showed increased insulin stimulation of 2-deoxyglucose uptake and glucose incorporation into glycogen compared with CHO-NEO, whereas Thr1134 receptors failed to signal these metabolic responses. We conclude that 1) transfected kinase-deficient insulin receptor mutants derived from insulin-resistant patients have distinct defects in the ability to mediate insulin action in vitro; 2) divergence of insulin signaling pathways may occur at the level of the receptor; and 3) normal activation of the receptor tyrosine kinase by insulin is not necessarily required for signaling of certain important biologic actions.     

Diacylglycerols modulate insulin action in rat adipocytes

Effect of 1,2-diacylglycerols on the insulin receptor function and insulin action in rat adipocytes was studied. 1,2-dioctanoylglycerol (100 micrograms/ml) did not alter insulin binding but it did stimulate phosphorylation of the beta-subunit of the insulin receptor as well as its tyrosine kinase activity. However, dioctanoylglycerol inhibited insulin-stimulated receptor autophosphorylation. This concentration of dioctanoylglycerol inhibited insulin-stimulated CO2 metabolism, lipogenesis and 3-O-methyl-glucose transport in a dose-dependent manner but did not alter any of these bioeffects in absence of insulin. While there was no direct link between diacylglycerol effect on tyrosine kinase activity of the insulin receptor and insulin action in rat adipocytes, the parallel inhibition of insulin-stimulated receptor autophosphorylation and insulin bioeffects by dioctanoylglycerol suggests its direct or indirect role in insulin signalling in rat fat cells.     

Mutations in the juxtamembrane region of the insulin receptor impair activation of phosphatidylinositol 3-kinase by insulin.

CHO/IRF960/T962 cells express a mutant human insulin receptor in which Tyr960 and Ser962 in the juxtamembrane region of the receptor's beta-subunit are replaced by Phe and Thr, respectively. The mutant insulin receptor undergoes autophosphorylation normally in response to insulin; however, insulin fails to stimulate thymidine incorporation into DNA, glycogen synthesis, and tyrosyl phosphorylation of an endogenous substrate pp185 in these cells. Another putative substrate of the insulin receptor tyrosine kinase is phosphatidylinositol 3-kinase (Ptdlns 3-kinase). We have previously shown that Ptdlns 3-kinase activity in Chinese hamster ovary cells expressing the wild-type human insulin receptor (CHO/IR) increases in both antiphosphotyrosine [anti-Tyr(P)] immunoprecipitates and intact cells in response to insulin. In the present study a new technique (detection of the 85-kDa subunit of Ptdlns 3-kinase using [32P]phosphorylated polyoma virus middle T-antigen as probe) is used to monitor the Ptdlns 3-kinase protein. The 85-kDa subunit of Ptdlns 3-kinase is precipitated by anti-Tyr(P) antibodies from insulin-stimulated CHO/IR cells, but markedly less protein is precipitated from CHO/IRF960/T962 cells. The amount of Ptdlns 3-kinase activity in the immunoprecipitates was also reduced in the CHO/IRF960/T962 cells compared to CHO/IR cells. In intact CHO/IRF960/T962 cells, insulin failed to stimulate phosphate incorporation into one of the products of activated Ptdlns 3-kinase, phosphatidylinositol-3,4-bisphosphate [Ptdlns(3,4)P2], whereas it caused a 12-fold increase in CHO/IR cells. In contrast, phosphate incorporation into another product, phosphatidylinositol trisphosphate [PtdlnsP3], was only partially depressed in the CHO/IRF960/T962 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro association of phosphatidylinositol 3-kinase activity with the activated insulin receptor tyrosine kinase.

We previously have shown that insulin treatment of cells greatly increases the activity of phosphatidylinositol (PI) 3-kinase in immunoprecipitates made with an antibody to phosphotyrosine. However, the association of PI 3-kinase activity with the activated insulin receptor is not significant under these conditions. In the present study, we have attempted to reconstitute the association of PI 3-kinase activity with the activated insulin receptor in vitro. PI 3-kinase activity does indeed associate with the autophosphorylated insulin receptor in our in vitro system. The autophosphorylation of the insulin receptor and/or its associated conformational change appear to be necessary for the association of PI 3-kinase activity with the receptor, since kinase negative receptor failed to bind PI 3-kinase activity. After binding, PI 3-kinase or its associated protein seems to be released from the activated receptor after the completion of its tyrosine phosphorylation by the receptor. Tyr960 in the juxtamembrane region of the insulin receptor beta-subunit seems to be involved in the association of PI 3-kinase activity with the receptor, but not C terminus region of the beta-subunit including two tyrosine autophosphorylation sites (Tyr1316 and Tyr1322). The in vitro assay system for the association of PI 3-kinase activity with the insulin receptor can be utilized to study the mechanism of interaction of these molecules and will be an useful method to detect other associated molecules with the insulin receptor.     

Intermolecular transphosphorylation between insulin receptors and EGF-insulin receptor chimerae.

The insulin receptor, a glycoprotein consisting of two extracellular alpha- and two transmembrane beta-subunits, is thought to mediate hormone action by means of its tyrosine-specific protein kinase activity. To explore the mechanism of insulin receptor phosphorylation we have used NIH3T3 cells transfected with two receptor constructs: one encoding a chimeric receptor composed of the extracellular domain of the human EGF receptor and the cytosolic domain of the human insulin receptor beta-subunit, and a second construct encoding a kinase-defiecient human insulin receptor. Stimulation of these cells with EGF induced tyrosine autophosphorylation of the EGF-insulin receptor chimera (150 kd) and tyrosine phosphorylation of the beta-subunit of the kinase-deficient insulin receptor (95 kd). The phosphopeptides of the autophosphorylated cytoplasmic domain of the EGF-insulin receptor chimera were comparable to those of the transphosphorylated beta-subunit of the kinase-deficient insulin receptor and of the wild-type human insulin receptor. When immunoaffinity purified EGF-insulin receptor hybrids and kinase-deficient insulin receptors were used in a cell lysate phosphorylation assay, it was found that addition of EGF produced 32P-labeling of both receptor species. We conclude that EGF acting directly through the EGF-insulin receptor chimera causes transphosphorylation of the kinase-deficient insulin receptor. These data support the notion that autophosphorylation of the insulin receptor may proceed by an intermolecular mechanism.     

Rat hepatoma cell variants resistant to insulin-diphtheria toxin A fragment conjugates. Genetic evidence for the separate pathways for insulin receptor-mediated mitogenic and hormonal stimulation

The rat hepatoma H35 cells in serum-free medium produce tyrosine aminotransferase (TAT) and initiate DNA synthesis and cell division upon exposure to 10(-9)-10(-10) M insulin. This insulin-dependent hormonal and mitogenic stimulation is through the insulin receptors and not through the receptors for insulin-like growth factor type I. We have isolated genetic variants of H35 cells which are resistant to a cytotoxic insulin-diphtheria toxin A fragment conjugate. These variants showed different degrees of insulin binding capacity and exhibited different sensitivities to insulin-stimulated TAT and DNA synthesis. Variant DTaI-b had a slight decrease in number of insulin receptors but completely lost sensitivity to both TAT and DNA stimulation. Variant 11-1 had a reduced number of insulin receptors but retained both TAT and DNA inducibilities. Variant 14-1, which had a high number of insulin receptors, was not responsive to DNA synthesis but was responsive to TAT induction. The beta-subunit of insulin receptors in these cell variants had different sensitivities to their insulin-dependent autophosphorylation. The rat hepatoma HTC cells used as a control showed very low insulin binding, no stimulation of TAT and DNA synthesis by insulin, and no detectable insulin-enhancement of beta-subunit phosphorylation. These characteristics provide genetic evidence for the divergence of the insulin receptor-mediated mitogenic and hormonal signals during the post-receptor pathways which is apparently regulated by the insulin-dependent phosphorylation.     

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.     

Phosphorylation and dephosphorylation of the insulin receptor: evidence against an intrinsic phosphatase activity

We have studied the reversibility of insulin receptor phosphorylation to establish the relation between this autophosphorylation reaction and the initiation of insulin action and between dephosphorylation and the termination of insulin effects in cells. In cultured Fao hepatoma cells labeled with 32PO4(3-), insulin increased 5-fold the phosphorylation of the beta-subunit of the insulin receptor at serine, threonine, and tyrosine residues. Addition of anti-insulin antiserum to cells incubated with insulin caused dissociation of insulin from the receptor and concurrent dephosphorylation of the beta-subunit. 32PO4(3-) associated with the insulin-stimulated receptor could be decreased by the addition of sodium phosphate to the medium but with a slower time course. Insulin stimulated phosphorylation of insulin receptor purified partially on immobilized wheat germ agglutinin. This reaction utilized [gamma-32P] ATP and occurred exclusively on tyrosine residues. Addition of unlabeled ATP caused a decrease in the amount of PO4(3-) associated with the receptor. Insulin-stimulated phosphorylation was also observed if the receptors were further purified by immunoprecipitation with anti-insulin receptor antibody prior to the phosphorylation reaction; however, addition of unlabeled ATP to this system did not chase the labeled 32PO4(3-) from the beta-subunit. These data are consistent with the notion that phosphorylation and dephosphorylation of the insulin receptor parallel the onset and termination of insulin action. Phosphatase activity involved in the dephosphorylation of the insulin receptor appears to be a glycoprotein because it was retained after partial purification of the receptor on wheat germ agglutinin-agarose; however, this phosphatase activity is distinct from the insulin receptor because it was not retained after immunoprecipitation of the receptor with anti-insulin receptor antibodies.     

Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes

Insulin stimulates hexose transport and phosphorylation of the insulin receptor in monolayer cultures of intact 3T3-L1 adipocytes. To assess the phosphorylation state of the receptor in situ, cells were equilibrated with [32P]orthophosphate and then disrupted under denaturing conditions which preserved the phosphorylation state of the receptor established in the cell. The insulin receptor, isolated by lectin adsorption and two-dimensional nonreducing/reducing polyacrylamide gel electrophoresis, occurred as a single oligomeric species with an apparent alpha 2 beta 2 subunit composition. This oligomeric structure was not altered by treating cells with insulin. Only the beta-subunit of the receptor was phosphorylated; [32P]phosphoserine and [32P] phosphotyrosine were both identified in the beta-subunit from cells in the unstimulated state, but only [32P] phosphotyrosine increased in cells stimulated with insulin. Neither insulin-like growth factors I nor II stimulated insulin receptor beta-subunit phosphorylation, although both activated hexose transport. Upon the addition of insulin, [32P]orthophosphate incorporated into the beta-subunit increased 4.5-fold (7-fold with respect to [32P]tyrosine) and was complete within 1 min (t1/2 = 8 s). Following the removal of insulin from the monolayers, [32P]beta-subunit fell to the basal level (t1/2 = 2.5 min); there was no lag phase before either transition. The tyrosine protein kinase activity, measured in vitro with a model substrate, was higher with immunoaffinity-purified insulin receptor from insulin-stimulated cells than from cells in the basal state. Hexose transport rate, measured using 3-O-[methyl-14C]glucose, was half-maximally stimulated at 2 nM insulin. A 1-min latency period followed insulin addition, after which a 7-fold increase in the steady-state rate of hexose uptake was achieved within 5 min. Upon the removal of insulin, hexose transport continued at the stimulated steady-state rate for 2.5 min and then declined to the basal rate with a half-time of 8 min. These kinetic experiments in situ and protein kinase activity measurements in vitro support the hypothesis that beta-subunit phosphorylation is an intermediate step linking insulin binding to the increased glucose transport rate.