Various proteins modulate the kinase activity of the insulin receptor

Previous studies of the substrate specificity of the purified insulin receptor tyrosine kinase using synthetic random polymers have demonstrated that the receptor kinase phosphorylates poly (Glu, Tyr) 4:1 but not poly (Glu, Tyr) 1:1. In the present study, insulin treatment of Chinese hamster ovary cells overexpressing the human insulin receptor was found to stimulate the ability of their membrane extracts to phosphorylate poly (Glu, Tyr) 1:1. It was concluded that this activity was due to the receptor itself because: 1) it was precipitated with a monoclonal antibody to the receptor; 2) the addition of various membrane extracts to purified insulin receptor preparations stimulated the ability of these preparations to phosphorylate poly (Glu, Tyr) 1:1; and 3) certain purified proteins, including bovine serum albumin and casein, were also capable of stimulating the purified receptor to phosphorylate poly (Glu, Tyr) 1:1. The effect of albumin was dose-dependent; 0.5 and 10 mg/ml bovine serum albumin stimulated the phosphorylation of poly (Glu, Tyr) 1:1 by 2- and 230-fold, respectively. In contrast, albumin had no effect on the phosphorylation of poly (Glu, Tyr) 4:1. These results indicate that the activity of the insulin receptor kinase on certain substrates can be modulated by the presence of other proteins.     

Phosphorylation of synthetic insulin receptor peptides by the insulin receptor kinase and evidence that the preferred sequence containing Tyr-1150 is phosphorylated in vivo

Three peptides were synthesized corresponding to potential autophosphorylation sites of the beta subunit of the human insulin receptor. These were peptide 1150 corresponding to amino acids 1142-1153 of the pro-receptor, peptide 960 corresponding to amino acids 952-961 of the proreceptor, and peptide 1316 corresponding to amino acids 1313-1329 of the proreceptor. Peptide 1150 served as a better substrate for the insulin receptor tyrosine protein kinase than either of the other peptides or than the Src peptide (corresponding to the sequence surrounding the autophosphorylation site at Tyr-416). Microsequencing of the phosphorylated peptide 1150 indicated that Tyr-1150 rather than Tyr-1146 or Tyr-1151 was phosphorylated in the in vitro reaction. The insulin receptor was then isolated from 32P-labeled IM-9 cells that had been exposed to insulin. Tryptic digestion of the beta subunit revealed one peptide whose phosphorylation was dependent upon insulin and occurred exclusively on Tyr. This peptide was selectively immunoprecipitated by an antipeptide antibody directed to the Tyr-1150-containing sequence. We conclude that Tyr-1150 is preferentially phosphorylated by the purified receptor kinase and that one of the autophosphorylation reactions elicited by insulin in intact cells occurs in a sequence that contains this residue.     

Insulin stimulation of the insulin receptor kinase can occur in the complete absence of beta subunit autophosphorylation

The glutamic acid:tyrosine (Glu:Tyr) synthetic polymer was observed to inhibit the insulin receptor beta subunit autophosphorylation with an IC50 of 0.20 mg/ml in the absence and 0.15 mg/ml in the presence of insulin. Even though complete blockade of beta subunit autophosphorylation was observed at 4.0 mg/ml Glu:Tyr, insulin was still capable of stimulating the exogenous protein kinase activity of the insulin receptor toward Glu:Tyr. Histone H2B (1.3 mg/ml) was also observed to inhibit the beta subunit autophosphorylation by approximately 80% with an IC50 of 0.31 and 0.35 mg/ml in the absence and presence of insulin, respectively. Similar to the results with Glu:Tyr, insulin was found to stimulate histone H2B phosphorylation under these conditions. Comparisons between the time courses of beta subunit autophosphorylation with those of Glu:Tyr phosphorylation both in the presence and absence of insulin confirmed that insulin can stimulate the exogenous protein kinase activity of the insulin receptor in the complete absence of beta subunit autophosphorylation. Prephosphorylation of the insulin receptor (from 0 to 1.3 mol of phosphate/mol of insulin receptor) in the absence of insulin was found to have no significant effect on the exogenous protein kinase activity when assayed both in the presence and absence of insulin. Insulin was observed to stimulate the phosphorylation of Glu:Tyr approximately 3-fold independent of the extent of beta subunit autophosphorylation. In contrast, prephosphorylation of the insulin receptors in the presence of insulin was observed to enhance the exogenous protein kinase activity dependent on the extent of autophosphorylation, such that by 1.4 mol of phosphate incorporated per mol of insulin receptor, insulin was found to maximally stimulate the initial rate of Glu:Tyr phosphorylation (approximately 9-fold). These results demonstrate that the insulin-dependent autophosphorylation of the insulin receptor results in an amplification of the insulin stimulation of the exogenous protein kinase activity, whereas the insulin-independent autophosphorylation does not.     

Identification of an autoinhibitory domain in the insulin receptor tyrosine kinase

We have tested the hypothesis that activation of the insulin receptor tyrosine kinase is due to autophosphorylation of tyrosines 1146, 1150 and 1151 within a putative autoinhibitory domain. A synthetic peptide corresponding to residues 1134–1162, with tyrosines substituted by alanine or phenylalanine, of the insulin receptor subunit was tested for its inhibitory potency and specificity towards the tyrosine kinase activity. This synthetic peptide gave inhibition of the insulin receptor tyrosine kinase autophosphorylation and phosphorylation of the exogenous substrate poly(Glu, Tyr) with an approximate IC₅₀ of 100 M. Inhibition appeared to be independent of the concentrations of insulin or the substrate poly(Glu, Tyr) but was decreased by increasing concentrations of ATP. This same peptide also inhibited the EGF receptor tyrosine kinase but not a serine/threonine protein kinase. These results are consistent with the hypothesis that this autophosphorylation domain contains an autoinhibitory sequence. (Mol Cell Biochem120: 103–110, 1993)Abbreviations IR Insulin Receptor - SDS/PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - CaM Calmodulin - HEPES 4-(2-Hydroxyethyl)-Piperazineethane-Sulfonic Acid - DMEM Dulbecco's Modified Eagle' Medium - PMSF Phenylmethyl-Sulfonyl Fluoride - HPLC High Performance Liquid Chromatography - PKC Protein Kinase C - PKI Inhibitory Peptide for cAMP-Kinase - CaMK II Ca²⁺/Calmodulin-Dependent Protein Kinase II - CaN A A Subunit of Calcineurin     

Characterization of Insulin-Like Growth Factor-I Receptors in PC 12 Pheochromocytoma Cells and Bovine Adrenal Medulla

Competitive binding studies indicated that PC12 cells have receptors for insulin-like growth factor-I (IGF-I). There are approximately 11,000 +/- 1,500 IGF-I receptors/cell; these receptors have an apparent KD for IGF-I of 7.2 +/- 0.6 nM. Covalent cross-linking of 125I-IGF-I to PC12 cells labeled a 125,000-130,000-Mr protein, presumably the alpha-subunit of the IGF-I receptor. Although PC12 cells also have insulin receptors, the 125I-IGF-I appeared to be cross-linked to IGF-I receptors, because 100 nM IGF-I competed for labeling but 100 nM insulin did not. Bovine chromaffin cells also have IGF-I receptors. The protein tyrosyl kinase activity of IGF-I receptors from bovine adrenal medulla and PC12 cells was examined after purification of the receptors by wheat germ agglutinin-Sepharose chromatography. IGF-I (10 nM) stimulated autophosphorylation of the beta-subunits of the IGF-I receptors from both preparations; the beta-subunits from both sources had Mr values of approximately 97,000. IGF-I also stimulated phosphorylation of the synthetic substrate poly(Glu:Tyr)4:1 by both receptor preparations. IGF-I (IC50 of approximately 0.2 nM) was much more potent than insulin at stimulating phosphorylation of poly(Glu:Tyr) by the bovine adrenal medulla preparation. A maximal concentration of IGF-I (10 nM) increased phosphorylation approximately threefold. IGF-I was slightly more effective than insulin at stimulating the phosphorylation of poly(Glu:Tyr) by the PC12 cell receptor preparation, but neither ligand produced a maximal effect at concentrations up to 100 nM. This result probably reflects the presence of comparable numbers of IGF-I and insulin receptors on PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinase inhibition by a phosphorylated peptide corresponding to the major insulin receptor autophosphorylation domain.

We studied the inhibitory effect of non-phosphorylated and triphosphorylated synthetic peptides, corresponding to amino acids 1143-1155 of the insulin proreceptor (domain 1151) on autophosphorylation and kinase of the insulin receptor. Tyrosine-phosphorylated peptides were synthesized using the N-(9-fluorenylmethoxycarbonyl)-O-dibenzylphosphono-L- tyrosine. The triphosphorylated peptide (1151-P3) and the non-phosphorylated peptide (1151-NP), respectively, inhibited insulin receptor autophosphorylation by 65% and 70%, in a dose-dependent and additive manner. When the receptor was pre-phosphorylated for 1 min with [gamma-32P]ATP, 1151-P3 decreased autophosphorylation to 60% of maximum, whereas 1151-NP had no further effect. In both non-activated and preactivated receptors, 1151-P3 inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu4, Tyr) was dose-dependently inhibited by both analogues. This effect was independent of the state of receptor phosphorylation or the addition of vanadate. Since 1151-P3 inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 1151-NP and 1151-P3 competition for the phosphorylation of a resin-immobilized 1151 peptide. While 1151-NP (at 2 mM) was highly competitive, inhibiting phosphate incorporation by 70%, 1151-P3 caused a four-fold increase in the phosphorylation of 1151-NP--resin. The receptor underwent conformational changes during autophosphorylation and an antibody directed against a peptide corresponding to amino acids 1314-1330 of the proreceptor (1322Ab) was previously shown to immunoprecipitate specifically the non-phosphorylated receptor forms. Nevertheless, the 1322Ab immunoprecipitated a fully autophosphorylated receptor in the presence of 1151-NP, but not of 1151-P3, thus suggesting a conformational change induced by the non-phosphorylated peptide. In conclusion, kinase inhibition was still observed after the addition of phosphate groups to three 1151-peptide tyrosines, but the peptide effect on receptor autophosphorylation, phosphorylation of homologous 1151-NP--resin and conformational changes induced in the receptor was altered dramatically. These data may provide a basis for further understanding the role of tyrosine phosphorylation in insulin receptor kinase activation or regulation.     

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.     

Lipid-induced insulin resistance in cultured hepatoma cells is associated with a decreased insulin receptor tyrosine kinase activity.

We have shown previously that experimental modifications of the cellular lipid composition of an insulin-sensitive rat hepatoma cell line (Zajdela Hepatoma Culture, ZHC) affect both binding and biological actions of insulin. Discrepancies between insulin binding and actions implied a postbinding defect, responsible for the observed insulin resistance in lipid-treated cells. To elucidate the mechanism for this defect, we have studied insulin binding and insulin receptor kinase activity in partially purified receptor preparations from ZHC cells grown either in normal medium or in medium supplemented with linoleic acid or 25-hydroxycholesterol. Insulin binding to the lectin-purified insulin receptor showed only a small alteration in receptor affinity for the preparations from lipid-treated cells. Insulin-stimulated autophosphorylation of the beta-subunit of the insulin receptor, as well as insulin-induced phosphorylation of the artificial substrate poly(Glu,Tyr)4:1, was significantly decreased in the preparations from lipid-modified cells. Although differences in basal levels were observed, the magnitude of the insulin-stimulated kinase activity was significantly decreased in receptor preparations from lipid-treated cells. These findings indicate that experimental modification of the lipids of cultured hepatoma cells can produce in insulin receptor kinase activity changes that are proportional to the reduced insulin action observed in these cells.     

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.     

Diacylglycerols modulate phosphorylation of the insulin receptor from human mononuclear cells

It has been found that 1,2- but not 1,3-diacylglycerols stimulated phosphorylation of the insulin receptor of cultured human monocyte-like (U-937) and lymphoblastoid (IM-9) cells both in the intact- and broken-cell systems. The stimulation of the receptor's beta-subunit phosphorylation was dose-dependent, with optimal effect at 100 micrograms/ml of diacylglycerol. The effects of insulin and 1,2-diacylglycerols on the phosphorylation of partially purified insulin receptors were additive. Phosphoamino acid analysis showed a major effect of diacylglycerols on phosphorylation of tyrosine residues. The diacylglycerols also stimulated tyrosine kinase activity of the partially purified U-937 and IM-9 insulin receptors 2.5-3.5-fold when measured by phosphorylation of an exogenous substrate, poly(Glu80Tyr20) in the absence of any added insulin, calcium or phospholipid. Since this diacylglycerol effect could not be reproduced under conditions optimal for protein kinase C activation and the purified protein kinase C did not stimulate phosphorylation of the beta-subunit of the insulin receptor in this system, it is unlikely that the diacylglycerol effect was mediated by protein kinase C. Since these exogenous 1,2-diacylglycerols at the same high concentration also inhibited 125I-insulin binding to the insulin receptor of the intact U-937 and IM-9 cells, diacylglycerols could modulate the function of the insulin receptor and insulin action in human mononuclear cells.     

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)     

Functional and immunological distinction between insulin-like growth factor I receptor subtypes in KB cells.

Subtypes of insulin-growth factor I (IGF-I) receptors, including hybrid receptors containing insulin receptor alpha beta dimers associated with IGF-I receptor alpha beta dimers, have been described in a number of systems. The molecular basis of the multiple subtypes and their functional significance is not understood. Ligand-dependent phosphorylation of insulin and IGF-I receptors and immunoprecipitation with antipeptide and monoclonal antibodies have been used to characterize the subpopulations of these receptors in the human KB cell line. IGF-I receptors exhibit beta subunits of 95 and 102 kDa in these cells. IGF-I receptors containing 102-kDa beta subunits are immunoprecipitated by the IGF-I receptor-specific antibody alpha-IR3. Antibody alpha-IR3 does not appear to recognize a hybrid receptor in these cells. However, an antipeptide antibody against the carboxyl-terminal domain of the insulin receptor (AbP5) immunoprecipitates a population of receptors phosphorylated in response to IGF-I (1 nM) which contains both 95- and 102-kDa beta subunits. These receptors must be hybrid complexes because AbP5 does not recognize the 102-kDa beta subunit directly. The inability of antibody alpha-IR3 to recognize these complexes suggests that their IGF-I receptor alpha subunits must differ from typical IGF-I receptor alpha subunits either in primary sequence or conformation. Therefore, KB cells may contain more than one type of IGF-I receptor alpha subunit. Hybrid IGF-I receptors can also be distinguished from homotypic IGF-I receptors by their responsiveness to IGF-II. Stimulation of autophosphorylation in hybrid IGF-I receptors by IGF-I is 3-4-fold greater than that seen in response to IGF-II. In contrast, IGF-I and IGF-II are nearly equipotent in stimulating autophosphorylation in the alpha-IR3-reactive receptor population. This suggests the existence of functionally distinct receptor subtypes which may differ in their ability to mediate the biological effects of IGF-II.     

Autophosphorylation within insulin receptor beta-subunits can occur as an intramolecular process.

The insulin receptor is a complex membrane-spanning glycoprotein composed of two alpha-subunits and two beta-subunits connected to form an alpha 2 beta 2 holoreceptor. Insulin binding to the extracellular alpha-subunits activates intracellular beta-subunit autophosphorylation and substrate kinase activity. The current study was designed to differentiate mechanisms of transmembrane signaling by the insulin receptor, specifically whether individual beta-subunits undergo cis- or trans-phosphorylation. We compared relative kinase activities of trypsin-truncated receptors, alpha beta-half receptors, and alpha 2 beta 2 holoreceptors under conditions that allowed us to differentiate intermolecular and intramolecular events. Compared to the insulin-stimulated holoreceptors, the trypsin-truncated receptor undergoes autophosphorylation at similar tyrosine residues and catalyzes substrate phosphorylation in the absence of insulin at a comparable rate. The truncated receptor sediments on a sucrose gradient at a position consistent with a structure comprising a single beta-subunit attached to a fragment of the alpha-subunit and undergoes autophosphorylation in this form in the absence of insulin. Autophosphorylation of the truncated insulin receptor is independent of receptor concentration, and immobilization of the truncated receptor on a matrix composed of an anti-receptor antibody bound to protein A-Sepharose diminishes neither autophosphorylation nor receptor-catalyzed substrate phosphorylation. Therefore, true intramolecular (cis) phosphorylations, which occur within individual beta-subunits derived from trypsin-truncated receptors, lead to kinase activation. However, insulin-stimulated autophosphorylation of insulin receptor alpha beta heterodimers is concentration-dependent, and both autophosphorylation and kinase activity are markedly reduced following immobilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of phosphatidylinositol kinase activity associated with the insulin receptor

Various lipids were tested as substrates for the insulin receptor kinase using either receptor partially purified from rat hepatoma cells by wheat-germ-agglutinin-Sepharose chromatography or receptor purified from human placenta by insulin-Sepharose affinity chromatography. Phosphatidylinositol was phosphorylated to phosphatidylinositol 4-phosphate by the partially purified insulin receptor. In contrast, phosphatidylinositol 4-phosphate and diacylglycerol were not phosphorylated. In some, but not all preparations of partially purified insulin receptor, the phosphatidylinositol kinase activity was stimulated by insulin (mean effect 33%). Phosphatidylinositol kinase activity was retained in insulin receptor purified to homogeneity. Insulin regulation of the phosphatidylinositol kinase was lost in the purified receptor; however, dithiothreitol stimulated both autophosphorylation of the purified receptor and phosphatidylinositol kinase activity in parallel about threefold. (Glu80Tyr20)n, a polymeric substrate specific to tyrosine kinases, inhibited the phosphatidylinositol kinase activity of the purified receptor by greater than 90% and inhibited receptor autophosphorylation by 67%. Immunoprecipitation by specific anti-receptor antibodies depleted by greater than 90% the phosphatidylinositol kinase activity in the supernatant of the purified receptor and the phosphatidylinositol kinase activity was recovered in the precipitate in parallel with receptor autophosphorylation activity. These characteristics of the phosphatidylinositol kinase activity of the purified insulin receptor and its metal ion preference paralleled those of the receptor tyrosine kinase activity and differed from bulk phosphatidylinositol kinase activity in cell extracts, which was not significantly inhibited by (Glu80Tyr20)n, stimulated by dithiothreitol or depleted by immunoprecipitation with anti-(insulin receptor) antibody. These results suggest that the insulin receptor is associated with a phosphatidylinositol kinase activity; however, this activity is not well regulated by insulin. This kinase appears to be distinct from the major phosphatidylinositol kinase(s) of cells. Its relationship to insulin action needs further study.     

Antibodies to insulin receptor tyrosine kinase stimulate its activity towards exogenous substrates without inducing receptor autophosphorylation.

Anti-peptide antibodies directed against a highly-conserved sequence of the insulin receptor tyrosine kinase domain have been used to study the relationship between this specific region and kinase activation. Antibodies have been prepared by the injection into a rabbit of a synthetic peptide (P2) corresponding to residues 1110-1125 of the proreceptor. The peptide exhibits 88-95% sequence similarity with the corresponding sequence in the v-ros protein and in receptors for epidermal growth factor and for insulin-like growth factor 1. Two antibodies with different specificities could be separated from total antiserum obtained after immunization with P2. One antibody [anti-(P-Tyr)] cross-reacted with phosphotyrosine and immunoprecipitated solely autophosphorylated receptors. This antibody was shown to increase or decrease the receptor tyrosine kinase activity depending on its concentration. In all circumstances receptor autophosphorylation and substrate phosphorylation were modulated in a parallel fashion. The second antibody (anti-P2) failed to immunoprecipitate the insulin receptor, but was found to interact with both the peptide and the receptor by e.l.i.s.a. assay. Using a tyrosine co-polymer we found that anti-P2 activated the insulin receptor kinase leading to substrate phosphorylation at a level similar to that observed with insulin. This effect was additive to the hormonal effect. In contrast, receptor autophosphorylation was not modified by the anti-peptide. The differential effect of this anti-peptide further supports the idea that receptor autophosphorylation and kinase activity towards exogenous substrates might be independently regulated. Finally, our data suggest that conformational changes in the receptor tyrosine kinase domain may be sufficient for activation of its enzymic activity.     

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.     

Decrease in beta-subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase.

In a previous study, we showed that the rat hepatic insulin receptor (IR) kinase of endosomes (ENs) was transiently activated to levels exceeding those of plasma membrane (PM) receptors following insulin injection. Phosphatase treatment of EN receptors abolished IR kinase activation implicating beta-subunit autophosphorylation as a mediator of the activation process (Khan, M. N., Baquiran, G., Brule, C., Burgess, J., Foster, B., Bergeron, J. J. M., and Posner, B. I. (1989) J. Biol. Chem. 264, 12931-12940). In the present study, the phosphotyrosine (PY) content of the IR beta-subunit in PM and ENs was estimated by two different methods. In one method, direct in vivo labeling with 32Pi followed by receptor immunoprecipitation was carried out. In the second method, immunoblotting with antibodies against the submembrane domain of the IR beta-subunit, encompassing residue 960 (alpha 960), and with antibodies against PY (alpha PY) was used to determine the content of PY/beta-subunit in PM and ENs following injection of insulin. By both methods, it was found that the PY content of PM IR was significantly greater than that of IR in ENs. With doses of 1.5 micrograms of insulin/100 g body weight (50% receptor occupancy) or 15 micrograms/100 g body weight (receptor saturation), the PY/beta-subunit of PM IR attained a level 2.0 to 2.5-fold of that observed for the IR of ENs. Surprisingly, the IR of ENs incorporated 3 to 5 times more PY/beta-subunit than those of PM consequent to autophosphorylation. Exogenous IR kinase activity (poly(Glu:Tyr)) in PM changed only slightly with insulin dose. In contrast, EN receptors exhibited a dose-dependent increase in kinase activity coincident with the decrease in PY/beta-subunit levels. A comparison of the proportion of receptor and kinase activity immunoprecipitated by alpha PY both before and after autophosphorylation indicated that ENs but not PM contained a small population of lightly phosphorylated but highly activated receptors. Since Thr12-Lys (IR kinase residues 1142-1153) efficiently inhibited IR autophosphorylation of both PM and EN receptors, Tris phosphorylation of beta-subunit regulatory tyrosines was unlikely. These results may be explicable by a dephosphorylation-dependent activation of IR kinase, as seen with the src family of tyrosine kinases.     

Insulin receptor kinase activity in rat liver. Regulation by fasting and high carbohydrate feeding

Insulin receptor kinase activity was measured in partially purified receptor preparations from livers of rats fed a standard diet or subjected to either prolonged fasting or a high carbohydrate (CHO) diet, conditions known to decrease (fasting) and increase (CHO) insulin action. Basal and insulin-stimulated phosphorylation of the beta subunit of the insulin receptor was comparable in all groups with a half-maximal effect at approximately 2.0 ng/ml free insulin and a 10-12-fold maximal effect. The kinase activity of insulin receptors from the three groups was further examined using the synthetic polypeptide Glu 4:Tyr 1. Basal and insulin-stimulated rates of Glu 4:Tyr 1 phosphorylation were highest in the CHO-fed and lowest in the fasted group. The magnitude of these differences was the same in the absence or presence of insulin; thus, the alterations in receptor kinase activity in fasting and CHO feeding were entirely expressed in the basal rate of peptide phosphorylation. Antireceptor antibody immunoprecipitated 70-80% of the basal Glu 4:Tyr 1 kinase activity in each group; the remaining 20-30% showed minor group differences when normalized for the amount of protein present in the receptor preparations. These results indicate that the group differences in basal kinase were intrinsic to the insulin receptor. Insulin increased the Vmax of Glu 4:Tyr 1 phosphorylation by approximately 30 fmol of phosphorus/fmol of binding activity/30 min in all three groups; however, the absolute Vmax was highest in the CHO-fed and lowest in the fasted group. The Km of Glu 4:Tyr 1 phosphorylation was unaffected by insulin and was comparable (approximately 0.25 mg/ml) in the three groups. These findings indicate that fasting and CHO feeding produce changes in receptor kinase activity which are regulated by mechanisms independent of insulin and that the alterations show substrate specificity so that differences are detected with one substrate (Glu 4:Tyr 1) but not another (the beta subunit).     

Autophosphorylation of the insulin receptor in vitro. Designation of phosphorylation sites and correlation with receptor kinase activation

Chemical degradation and antipeptide antibodies were used to study alterations in the structure and function of the human placental insulin receptor following autophosphorylation in vitro. Antibodies elicited to residues 1143-1162 (P2) of the human insulin proreceptor immunoprecipitated the native, phosphorylated receptor but not the unphosphorylated receptor. Since this antibody recognizes both forms of the receptor on immunoblots, it was concluded that the accessibility of the P2 domain to the antibody is increased by in vitro autophosphorylation. Chemical cleavage at either tryptophan or methionine residues followed by immunoprecipitation with antipeptide antibodies was used to map the in vitro autophosphorylation sites of the beta subunit of the insulin receptor. Two phosphorylated fragments were resolved. One, recognized by antibody elicited to amino acid residues 1328-1343 (P5), is derived from the carboxyl terminus of the beta subunit and includes tyrosine 1316. The other, recognized by antibody to P2, is located in a domain that includes tyrosine 1150. The rate of phosphorylation of this latter site correlates with the rate of activation of the insulin receptor kinase during in vitro autophosphorylation. The results support the following conclusions: autophosphorylation alters the conformation of the beta subunit of the insulin receptor; autophosphorylation in vitro leads to phosphorylation of tyrosine residues near the carboxyl terminus of the protein and in the P2 domain that includes tyrosine 1150; activation of the insulin receptor kinase correlates with autophosphorylation of the domain containing tyrosine 1150.     

Increased protein kinase C activity is linked to reduced insulin receptor autophosphorylation in liver of starved rats

Phosphorylation of the insulin receptor beta-subunit on serine/threonine residues by protein kinase C reduces both receptor kinase activity and insulin action in cultured cells. Whether this mechanism regulates insulin action in intact animals was investigated in rats rendered insulin-resistant by 3 days of starvation. Insulin-stimulated autophosphorylation of the partially purified hepatic insulin receptor beta-subunit was decreased by 45% in starved animals compared to fed controls. This autophosphorylation defect was entirely reversed by removal of pre-existing phosphate from the receptor with alkaline phosphatase, suggesting that increased basal phosphorylation on serine/threonine residues may cause the decreased receptor tyrosine kinase activity. Tryptic removal of a C-terminal region of the receptor beta-subunit containing the Ser/Thr phosphorylation sites similarly normalized receptor autophosphorylation. To investigate which kinase(s) may be responsible for such increased Ser/Thr phosphorylation in vivo, protein kinase C and cAMP-dependent protein kinase A in liver were studied. A 2-fold increase in protein kinase C activity was found in both cytosol and membrane extracts from starved rats as compared to controls, while protein kinase A activity was diminished in the cytosol of starved rats. A parallel increase in protein kinase C was demonstrated by immunoblotting with a polyclonal antibody which recognizes several protein kinase C isoforms. These findings suggest that in starved, insulin-resistant animals, an increase in hepatic protein kinase C activity is associated with increased Ser/Thr phosphorylation which in turn decreases autophosphorylation and function of the insulin receptor kinase.