Protein tyrosine phosphatase 1B interacts with the activated insulin receptor.

Protein tyrosine phosphatase 1B (PTP1B) is a protein tyrosine phosphatase of unknown function, although increasing evidence supports a role for this phosphatase in insulin action. We have investigated the interaction of PTP1B with the insulin receptor using a PTP1B glutathione S-transferase (GST) fusion protein with a point mutation in the enzyme's catalytic domain. This fusion protein is catalytically inactive, but the phosphatase's phosphotyrosine binding site is maintained. The activated insulin receptor was precipitated from purified receptor preparations and whole-cell lysates by the inactive PTP1B-GST, demonstrating a direct association between the insulin receptor and PTP1B. A p120 of unknown identity was also precipitated from whole-cell lysates by the PTP1B fusion protein, but IRS-1 (pp185) was not. A catalytically inactive [35S]PTP1B-fusion protein bound directly to immobilized insulin receptor kinase domains and was displaced in a concentration-dependent manner. Finally, tyrosine-phosphorylated PTP1B was precipitated from whole-cell lysates by an anti-insulin receptor antibody after insulin stimulation. The site of interaction between PTP1B and the insulin receptor was studied using phosphopeptides modeled after the receptor's kinase domain, the NPXY domain, and the COOH-terminal. Each phosphopeptide inhibited the PTP1B-GST:insulin receptor interaction. Study of mutant insulin receptors demonstrated that activation of the kinase domain is necessary for the PTP1B:insulin receptor interaction, but receptors with deletion of the NPXY domain or of the COOH-terminal can still bind to the PTP1B-GST. We conclude that PTP1B can associate directly with the activated insulin receptor at multiple different phosphotyrosine sites and that dephosphorylation by PTP1B may play a significant role in insulin receptor signal transduction.

Oxygen consumption and carbon dioxide production in male prairie deermice (Peromyscus maniculatus bairdii) in different reproductive conditions and group densities.

Natural and laboratory populations of Peromyscus exhibit a profound but reversible reproductive inhibition related to population density. Our earlier studies described the endocrine physiology of inhibited animals which resembles a condition of delayed puberty, but they did not reveal a primary mechanism for the induction and maintenance of the inhibition. These studies indicated that reproductive inhibition could be associated with an overall change in general metabolism. To test this hypothesis, oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured in three groups of Peromyscus maniculatus males that were: 1) reproductively-proven, 2) reproductively-inhibited, or 3) recovered from inhibition. We found that the mean of the 2-hr period with the lowest VO2 (the Resting Metabolic Rate, or RMR) was significantly lower, and the mean Respiratory Exchange Ratio (RER) was significantly higher in reproductively-inhibited males compared with reproductively-proven males. In addition, previously inhibited males allowed to recover reproductive function had a significantly higher mean VO2, while the mean RER was not different from reproductively-proven males. Moreover, and contrary to some studies with other species, increasing the ambient carbon dioxide concentration or the caging densities to as high as six animals did not significantly affect oxygen consumption when compared with the corresponding values for individuals. Taken together, these findings indicate that the reproductive inhibition observed in P. maniculatus laboratory populations is causally associated with a significant reduction in general metabolism and that this metabolic reduction which is associated with reproductive-inhibition is not induced by a CO2 signal or induced by absolute density, per se.

Platelet-derived growth factor inhibits insulin stimulation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase in 3T3-L1 adipocytes without affecting glucose transport.

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for insulin-responsive glucose transporter (GLUT4) translocation and glucose transport. Insulin and platelet-derived growth factor (PDGF) stimulate PI3K activity in 3T3-L1 adipocytes, but only insulin is capable of stimulating GLUT4 translocation and glucose transport. We found that PDGF causes serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) in 3T3-L1 cells, measured by altered mobility on SDS-polyacrylamide gel, and this leads to a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1. The PI3K inhibitors wortmannin and LY294002 inhibit the PDGF-induced phosphorylation of IRS-1, whereas the MEK inhibitor PD98059 was without a major effect. PDGF pretreatment for 60-90 min led to a marked 80-90% reduction in insulin stimulatable phosphotyrosine and IRS-1-associated PI3K activity. We examined the functional consequences of this decrease in IRS-1-associated PI3K activity. Interestingly, insulin stimulation of GLUT4 translocation and glucose transport was unaffected by 60-90 min of PDGF preincubation. Furthermore, insulin activation of Akt and p70(s6kinase), kinases downstream of PI3K, was unaffected by PDGF pretreatment. Wortmannin was capable of blocking these insulin actions following PDGF pretreatment, suggesting that PI3K was still necessary for these effects. In conclusion, 1) PDGF causes serine/threonine phosphorylation of IRS-1, and PI3K, or a kinase downstream of PI3K, mediates this phosphorylation. 2) This PDGF-induced phosphorylation of IRS-1 leads to a significant decrease in insulin-stimulated PI3K activity. 3) PDGF has no effect on insulin stimulation of Akt, p70(s6kinase), GLUT4 translocation, or glucose transport. 4) This suggests the existence of an IRS-1-independent pathway leading to the activation of PI3K, Akt, and p70(s6kinase); GLUT4 translocation; and glucose transport.

Localization of the insulin-like growth factor I receptor binding sites for the SH2 domain proteins p85, Syp, and GTPase activating protein.

Potential signaling substrates for the insulin-like growth factor I (IGF-I) receptor are SH2 domain proteins including the p85 subunit of phosphatidylinositol 3-kinase, the tyrosine phosphatase Syp, GTPase activating protein (GAP), and phospholipase C-gamma (PLC-gamma). In this study, we demonstrate an association between the IGF-I receptor and p85, Syp, and GAP, but not with PLC-gamma in lysates of cells overexpressing the human IGF-I receptor. We further investigated these interactions using glutathione S-transferase (GST) fusion proteins containing the amino-terminal SH2 domains of p85 or GAP, or both SH2 domains of Syp or PLC-gamma to precipitate the IGF-I receptor from purified receptor preparations and from whole cell lysates. p85-, Syp-, and GAP-GSTs precipitated the IGF-I receptor, whereas the PLC-gamma-GST did not. Using phosphopeptides corresponding to IGF-I receptor phosphorylation sites, we determined that the p85- and Syp-GST association with the IGF-I receptor could be inhibited by a carboxyl-terminal peptide containing pY1316 and that the GAP-GST association could be inhibited by a NPXY domain peptide. The GAP-GST binding site was confirmed by showing that a mutant IGF-I receptor with a deletion of the NPXY domain including tyrosine 950 was poorly precipitated by the GAP-GST. We conclude that p85 and Syp may bind directly to the IGF-I receptor at tyrosine 1316, and that GAP may bind to the IGF-I receptor at and PLC-gamma was not evident. p85, Syp, and GAP are potential modulators of IGF-I receptor signal transduction.

Localization of the insulin receptor binding sites for the SH2 domain proteins p85, Syp, and GAP.

The insulin receptor is known to interact with the SH2 domain proteins p85 (the regulatory subunit of phosphatidylinositol 3-kinase), Syp (a tyrosine phosphatase), and GAP (GTPase-activating protein). In this study, we mapped the insulin receptor binding sites for each of these proteins by examining the ability of phosphopeptides, corresponding to insulin receptor phosphorylation sites, and mutant insulin receptors to inhibit an insulin receptor-SH2 domain interaction. Precipitation of partially purified insulin receptors by glutathione S-transferase fusion proteins containing the N-terminal SH2 domains of p85 and GAP and both SH2 domains of Syp was demonstrated. The effect of the addition of each phosphopeptide on insulin receptor precipitation was tested. pY1322, the C-terminal insulin receptor peptide, inhibited insulin receptor precipitation by both p85- and Syp-GST. The NPXY internalization domain peptide inhibited insulin receptor precipitation by GAP-GST. These data were confirmed by mutant insulin receptor experiments. The insulin receptor C-terminal mutants, delta CT and Y/F2, were not precipitated by p85- or Syp-GST and the NPXY mutant insulin receptors, delta Ex16 and HI delta NPEY, were not precipitated by GAP-GST. Therefore, we conclude that p85 and Syp bind to the insulin receptor C terminus at tyrosine 1322 and GAP binds to the insulin receptor NPXY domain at tyrosine 960.