The Experts below are selected from a list of 177 Experts worldwide ranked by ideXlab platform
Atsunori Kashiwagi - One of the best experts on this subject based on the ideXlab platform.
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Insulin Receptor Kinase phosphorylates protein tyrosine phosphatase containing Src homology 2 regions and modulates its PTPase activity in vitro
Biochemical and biophysical research communications, 1994Co-Authors: Hiroshi Maegawa, Satoshi Ugi, Ryuichi Kikkawa, Yukio Shigeta, Masaaki Adachi, Yuji Hinoda, Akira Yachi, Atsunori KashiwagiAbstract:To clarify the role of protein tyrosine phosphatase (PTPase) containing a pair of Src homology 2 (SH2) regions upon Insulin signaling, we studied the interactions between the Insulin Receptor and SH-PTP2 coupled to glutathione-S-transferase. A full length SH-PTP2 was phosphorylated by Insulin Receptor Kinase and associated with the Insulin Receptor in vitro. The N-terminal SH2 domain was more phosphorylated than the other SH2 domain of SH-PTP2. However, both SH2 domains of SH-PTP2 were necessary for association with Insulin Receptors. Phosphorylation of the SH2 domains of SH-PTP2 resulted in decreased PTPase activities toward the phosphorylated Insulin Receptor. These results indicate that the Insulin Receptor can negatively regulate SH-PTP2 activity by means of phosphorylating the SH2 domains.
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Pioglitazone Ameliorates High Glucose Induced Desensitization of Insulin Receptor Kinase in Rat 1 Fibroblasts in Culture
Biochemical and biophysical research communications, 1993Co-Authors: Hiroshi Maegawa, R. Tachikawaide, Satoshi Ugi, M. Iwanishi, K. Egawa, Ryuichi Kikkawa, Yukio Shigeta, Atsunori KashiwagiAbstract:Abstract A new oral agent, pioglitazone, increases Insulin sensitivity by activating Receptor Kinase in Insulin-resistant rats. To clarify the mechanism, we studied in vitro effects of glucose and pioglitazone on the Insulin Receptor function using Rat 1 fibroblasts which expressed human Insulin Receptors. Insulin Receptor Kinase activity was impaired by incubating cells for 4 days in the presence of 27mM D-glucose. The glucose effect was time- and dose-dependent and also specific for D-glucose, since D-raffinose incubation had no effect. Pioglitazone treatment did not have any effect on intact Receptor Kinase. However, exposure of both 27mM D-glucose and 0.1 μ M pioglitazone to the cells completely prevented the glucose-induced impairment of Insulin Receptor Kinase activity, suggesting that pioglitazone might reverse the processes which are critical for the glucose-induced desensitization of Insulin Receptor Kinase.
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Src homology 2 domains of protein tyrosine phosphatase are phosphorylated by Insulin Receptor Kinase and bind to the COOH-terminus of Insulin Receptors in vitro
Biochemical and biophysical research communications, 1993Co-Authors: Hiroshi Maegawa, R. Tachikawaide, Satoshi Ugi, Ryuichi Kikkawa, Yukio Shigeta, O. Ishibashi, N. Takahara, Yasushi Tanaka, Y. Takagi, Atsunori KashiwagiAbstract:Abstract To clarify the role of protein tyrosine phosphatases(PTPase) containing Src homology 2 (SH2) regions on Insulin signaling, we investigated the interactions between SH2 regions of PTPase and Insulin Receptors. We made a pair of SH2 domains of PTP1C and SH-PTP2 fusion proteins coupled to glutathione-S-transferase (GST) using pGEX-3X expression vector. After incubating with Insulin, Insulin Receptors were incubated with SH2 proteins in the presence of 100 μ ATP at 4°C for 3 hr, and then immunoprecipitated and analyzed by SDS-PAGE. We found that SH2 domains of SH-PTP2 were phosphorylated, but not those of PIP1C by Insulin Receptor Kinase and the SH2 domains of SH-PTP2, but not those of PTP1C, directly bound to the phosphorylated COOH-terminus of Insulin Receptors in vitro .
Morris F. White - One of the best experts on this subject based on the ideXlab platform.
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Insulin Receptor Kinase domain autophosphorylation regulates Receptor enzymatic function.
The Journal of biological chemistry, 1992Co-Authors: P A Wilden, K Siddle, C R Kahn, Morris F. WhiteAbstract:Abstract 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.
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The role of Insulin Receptor Kinase domain autophosphorylation in Receptor-mediated activities. Analysis with Insulin and anti-Receptor antibodies.
The Journal of biological chemistry, 1992Co-Authors: P A Wilden, Morris F. White, K Siddle, E Haring, Jonathan M. Backer, C R KahnAbstract:Abstract 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.
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YMXM motifs of IRS-1 define substrate specificity of the Insulin Receptor Kinase
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Steven E. Shoelson, Swati Chatterjee, Manas Chaudhuri, Morris F. WhiteAbstract:Abstract Of 34 tyrosine residues in Insulin Receptor substrate 1 (IRS-1), 14 are adjacent to acidic residues, suggesting that they might be phosphorylation sites. Synthetic peptides corresponding to sequences surrounding these tyrosines were used as substrates of the Insulin Receptor Kinase. Surprisingly six of these, each within YMXM motifs, were phosphorylated with greatest efficiency (Km, 24-92 microM; kcat/Km, 0.6-2.1 x 10(4) M-1.sec-1). Substituted YMXM peptides revealed a strong preference of the Insulin Receptor Kinase for methionine at Y + 1 and Y + 3 positions. When phosphorylated, related YMXM sequences are recognition motifs for binding to proteins with src-homology (SH2) domains. The combined hydrophobic and flexible nature of methionine side chains adjacent to the targeted tyrosines provides a versatile contact for recognition by diverse proteins involved in signal transduction.
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Pertussis toxin inhibits autophosphorylation and activation of the Insulin Receptor Kinase.
Biochemical and biophysical research communications, 1991Co-Authors: Dirk Müller-wieland, Morris F. White, Wilhelm Krone, Bert Behnke, Angelika Gebhardt, Sandra Neumann, C R KahnAbstract:Pertussis toxin is an ADP-ribosyltransferase which alters the function of some of the GTP-binding proteins and inhibits some actions of Insulin. In vivo, pertussis toxin (2 micrograms/ml/2h) inhibited Insulin-stimulated tyrosyl autophosphorylation of the Insulin Receptor by 50% in FaO cells, and nearly completely inhibited phosphorylation of the cellular Insulin Receptor substrate pp185. Similarly, Insulin-stimulated autophosphorylation and Kinase activity of the Insulin Receptor purified on wheat germ agglutinin-agarose from pertussis toxin-treated FaO cells was diminished 50%; however, treatment of cells with the catalytically inactive B-oligomer of the toxin had no effect on Receptor tyrosine Kinase activity in vitro. Pertussis toxin did not alter Insulin binding or the cellular levels of ATP, cAMP, and cGMP. Furthermore, immunoprecipitation of the Insulin Receptor from intact cells with anti-Insulin Receptor antibodies showed that pertussis toxin did not increase the phosphorylation of serine or threonine residues in the Insulin Receptor. These results suggest that pertussis toxin can modulate signal transduction of Insulin at the level of the Insulin Receptor Kinase.
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structure of the Insulin Receptor substrate irs 1 defines a unique signal transduction protein
Nature, 1991Co-Authors: Xiao Jian Sun, Paul L. Rothenberg, P A Wilden, Jonathan M. Backer, Ronald C Kahn, E Araki, D A Cahill, Barry J Goldstein, Morris F. WhiteAbstract:SINCE the discovery of Insulin nearly 70 years ago, there has been no problem more fundamental to diabetes research than understanding how Insulin works at the cellular level. Insulin binds to the α subunit of the Insulin Receptor which activates the tyrosine Kinase in the β subunit, but the molecular events linking the Receptor Kinase to Insulin-sensitive enzymes and transport processes are unknown1,2. Our discovery that Insulin stimulates tyrosine phosphorylation of a protein of relative molecular mass between 165,000 and 185,000, collectively called pp185, showed that the Insulin Receptor Kinase has specific cellular substrates3. The pp185 is a minor cytoplasmic phosphoprotein found in most cells and tissues4–10; its phosphorylation is decreased in cells expressing mutant Receptors defective in signalling6,11. We have now cloned IRS-1, which encodes a component of the pp185 band. IRS-1 contains over ten potential tyrosine phosphorylation sites, six of which are in Tyr-Met-X-Met motifs. During Insulin stimulation, the IRS-1 protein undergoes tyrosine phosphorylation and binds phosphatidylinositol 3-Kinase, suggesting that IRS-1 acts as a multisite Mocking' protein to bind signal-transducing molecules containing Src-homology 2 and Src-homology-3 domains12–14. Thus IRS–1 may link the Insulin Receptor Kinase and enzymes regulating cellular growth and metabolism.
Hiroshi Maegawa - One of the best experts on this subject based on the ideXlab platform.
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Insulin Receptor Kinase phosphorylates protein tyrosine phosphatase containing Src homology 2 regions and modulates its PTPase activity in vitro
Biochemical and biophysical research communications, 1994Co-Authors: Hiroshi Maegawa, Satoshi Ugi, Ryuichi Kikkawa, Yukio Shigeta, Masaaki Adachi, Yuji Hinoda, Akira Yachi, Atsunori KashiwagiAbstract:To clarify the role of protein tyrosine phosphatase (PTPase) containing a pair of Src homology 2 (SH2) regions upon Insulin signaling, we studied the interactions between the Insulin Receptor and SH-PTP2 coupled to glutathione-S-transferase. A full length SH-PTP2 was phosphorylated by Insulin Receptor Kinase and associated with the Insulin Receptor in vitro. The N-terminal SH2 domain was more phosphorylated than the other SH2 domain of SH-PTP2. However, both SH2 domains of SH-PTP2 were necessary for association with Insulin Receptors. Phosphorylation of the SH2 domains of SH-PTP2 resulted in decreased PTPase activities toward the phosphorylated Insulin Receptor. These results indicate that the Insulin Receptor can negatively regulate SH-PTP2 activity by means of phosphorylating the SH2 domains.
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Pioglitazone Ameliorates High Glucose Induced Desensitization of Insulin Receptor Kinase in Rat 1 Fibroblasts in Culture
Biochemical and biophysical research communications, 1993Co-Authors: Hiroshi Maegawa, R. Tachikawaide, Satoshi Ugi, M. Iwanishi, K. Egawa, Ryuichi Kikkawa, Yukio Shigeta, Atsunori KashiwagiAbstract:Abstract A new oral agent, pioglitazone, increases Insulin sensitivity by activating Receptor Kinase in Insulin-resistant rats. To clarify the mechanism, we studied in vitro effects of glucose and pioglitazone on the Insulin Receptor function using Rat 1 fibroblasts which expressed human Insulin Receptors. Insulin Receptor Kinase activity was impaired by incubating cells for 4 days in the presence of 27mM D-glucose. The glucose effect was time- and dose-dependent and also specific for D-glucose, since D-raffinose incubation had no effect. Pioglitazone treatment did not have any effect on intact Receptor Kinase. However, exposure of both 27mM D-glucose and 0.1 μ M pioglitazone to the cells completely prevented the glucose-induced impairment of Insulin Receptor Kinase activity, suggesting that pioglitazone might reverse the processes which are critical for the glucose-induced desensitization of Insulin Receptor Kinase.
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Src homology 2 domains of protein tyrosine phosphatase are phosphorylated by Insulin Receptor Kinase and bind to the COOH-terminus of Insulin Receptors in vitro
Biochemical and biophysical research communications, 1993Co-Authors: Hiroshi Maegawa, R. Tachikawaide, Satoshi Ugi, Ryuichi Kikkawa, Yukio Shigeta, O. Ishibashi, N. Takahara, Yasushi Tanaka, Y. Takagi, Atsunori KashiwagiAbstract:Abstract To clarify the role of protein tyrosine phosphatases(PTPase) containing Src homology 2 (SH2) regions on Insulin signaling, we investigated the interactions between SH2 regions of PTPase and Insulin Receptors. We made a pair of SH2 domains of PTP1C and SH-PTP2 fusion proteins coupled to glutathione-S-transferase (GST) using pGEX-3X expression vector. After incubating with Insulin, Insulin Receptors were incubated with SH2 proteins in the presence of 100 μ ATP at 4°C for 3 hr, and then immunoprecipitated and analyzed by SDS-PAGE. We found that SH2 domains of SH-PTP2 were phosphorylated, but not those of PIP1C by Insulin Receptor Kinase and the SH2 domains of SH-PTP2, but not those of PTP1C, directly bound to the phosphorylated COOH-terminus of Insulin Receptors in vitro .
Ramji L. Khandelwal - One of the best experts on this subject based on the ideXlab platform.
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Does the Insulin-mimetic action of vanadate involve Insulin Receptor Kinase?
Molecular and Cellular Biochemistry, 1993Co-Authors: Subbiah Pugazhenthi, Ramji L. KhandelwalAbstract:Effects of vanadate administration on the Insulin Receptor status in liver were examined in streptozotocin-induced diabetic rats. Diabetic rats were characterized by hyperglycemia (4-fold increase), hypoInsulinemia (81% decrease) and a significant (P
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Does the Insulin-mimetic action of vanadate involve Insulin Receptor Kinase?
Molecular and cellular biochemistry, 1993Co-Authors: Subbiah Pugazhenthi, Ramji L. KhandelwalAbstract:Effects of vanadate administration on the Insulin Receptor status in liver were examined in streptozotocin-induced diabetic rats. Diabetic rats were characterized by hyperglycemia (4-fold increase), hypoInsulinemia (81% decrease) and a significant (P < 0.01) increase in hepatic Insulin Receptor numbers. Autophosphorylation of the beta subunit of Insulin Receptor and its tyrosine Kinase activity towards the synthetic peptide (poly glut4tyr1) decreased by approximately 60% as a result of diabetes. After chronic treatment of these rats with sodium orthovanadate, the plasma glucose levels were normalized to near control values with the hypoInsulinemia remaining unaltered. The Insulin-stimulated phosphorylation of the beta subunit increased significantly (P < 0.001) in diabetic rats after treatment with vanadate. However, the improvement in the tyrosine Kinase activity was marginal. In vitro, vanadate prevented the dephosphorylation of the phosphorylated Insulin Receptor and increased its tyrosine Kinase activity in the absence as well as presence of Insulin. The findings of this study further support the view that Insulin Receptor is one of the sites involved in the Insulin-mimetic actions of vanadate.
P A Wilden - One of the best experts on this subject based on the ideXlab platform.
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Insulin Receptor Kinase domain autophosphorylation regulates Receptor enzymatic function.
The Journal of biological chemistry, 1992Co-Authors: P A Wilden, K Siddle, C R Kahn, Morris F. WhiteAbstract:Abstract 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.
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The role of Insulin Receptor Kinase domain autophosphorylation in Receptor-mediated activities. Analysis with Insulin and anti-Receptor antibodies.
The Journal of biological chemistry, 1992Co-Authors: P A Wilden, Morris F. White, K Siddle, E Haring, Jonathan M. Backer, C R KahnAbstract:Abstract 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.
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The Role of Insulin Receptor Kinase Domain Autophosphorylation in Receptor-mediated Activities
1992Co-Authors: P A Wilden, E Haring, Jonathan M. Backer, Kenneth Siddles, Morris F. Whitell, C R KahnAbstract:The role of specific tyrosine autophosphorylation sites in the human Insulin Receptor Kinase domain (TyrllSS, Tyr116’, and T~r’l~~) 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 tripleSer 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 halfmaximal 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 pp185/IRS-1. Interestingly, all mutants showed a marked decrease in Insulin-stimulated internalization. AntiReceptor 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
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structure of the Insulin Receptor substrate irs 1 defines a unique signal transduction protein
Nature, 1991Co-Authors: Xiao Jian Sun, Paul L. Rothenberg, P A Wilden, Jonathan M. Backer, Ronald C Kahn, E Araki, D A Cahill, Barry J Goldstein, Morris F. WhiteAbstract:SINCE the discovery of Insulin nearly 70 years ago, there has been no problem more fundamental to diabetes research than understanding how Insulin works at the cellular level. Insulin binds to the α subunit of the Insulin Receptor which activates the tyrosine Kinase in the β subunit, but the molecular events linking the Receptor Kinase to Insulin-sensitive enzymes and transport processes are unknown1,2. Our discovery that Insulin stimulates tyrosine phosphorylation of a protein of relative molecular mass between 165,000 and 185,000, collectively called pp185, showed that the Insulin Receptor Kinase has specific cellular substrates3. The pp185 is a minor cytoplasmic phosphoprotein found in most cells and tissues4–10; its phosphorylation is decreased in cells expressing mutant Receptors defective in signalling6,11. We have now cloned IRS-1, which encodes a component of the pp185 band. IRS-1 contains over ten potential tyrosine phosphorylation sites, six of which are in Tyr-Met-X-Met motifs. During Insulin stimulation, the IRS-1 protein undergoes tyrosine phosphorylation and binds phosphatidylinositol 3-Kinase, suggesting that IRS-1 acts as a multisite Mocking' protein to bind signal-transducing molecules containing Src-homology 2 and Src-homology-3 domains12–14. Thus IRS–1 may link the Insulin Receptor Kinase and enzymes regulating cellular growth and metabolism.