The focal adhesion kinase p125Fak is a widely expressed cytosolic tyrosine kinase, which is involved in integrin signaling and in signal transduction of a number of growth factors. In contrast to tyrosine kinase receptors such as the platelet-derived growth factor and the hepatocyte growth factor receptors, which induce p125Fak phosphorylation, insulin has been shown to promote its dephosphorylation. Fak is dependent on the cell architecture, and hence the interaction of the insulin/IGF-I signaling system with the integrin system will vary accordingly. The focal adhesion kinase p125Fak is a cytosolic tyrosine kinase initially isolated from Src-transformed cells (1, 2). It is localized at focal adhesion plaques of cultured cells and binds to a number of proteins involved in the organization of the cytoskeleton and to signaling molecules, resulting in the formation of multicomponents complexes (reviewed in Refs. 3-6). Tyrosine phosphorylation of p125Fak occurs rapidly in response to integrin clustering or binding to the extracellular matrix, and this correlates with increased kinase activity (2, 7-9). For most cell types, cooperation of adhesion-mediated and growth factor-mediated signaling pathways is required for appropriate growth control, and it is now widely accepted that p125Fak may be a point of convergence in the actions of integrins and growth factors (10, 11). Indeed, p125Fak is not only activated by integrins, but also by mitogenic neuropeptides (12), thrombin (13), sphingosine (14), the bioactive lipid lysophosphatidic acid (15-17), and by ligands for tyrosine kinase receptors such as platelet-derived growth factor and hepatocyte growth factor (18 -20).In contrast to other tyrosine kinase receptors, which induce tyrosine phosphorylation of p125Fak , it has been reported that in fibroblasts insulin promotes a decrease in p125Fak phosphorylation (21-23).The insulin receptor is a heterotetrameric oligomer consisting of two extracellular 135-kDa ␣-subunits and two 95-kDa transmembrane ␤-subunits containing a tyrosine kinase (24,25). Insulin binding to the ␣-subunit stimulates autophosphorylation of the ␤-subunit cytoplasmic domain on multiple tyrosine residues. This activates the receptor kinase, leading to phosphorylation of several substrates including IRS-1, 1 IRS-2, Shc, and Gab-1 (26 -31). IRS-1/2 and Gab-1 carry multiple potential tyrosine phosphorylation sites, which upon phosphorylation become docking sites for SH2 domain-containing proteins. These include the p85 regulatory subunit of phosphatidylinositol 3-kinase, Grb2, and the phosphotyrosine phosphatase SHP-2 (32-34).IRS-1 is also found to be associated with Csk, the C-terminal Src kinase that is an inhibitor of the Src kinase family. It has been shown recently that the insulin-induced complex of IRS-1 and Csk could be involved in dephosphorylation of p125 Fak observed after insulin treatment of fibroblasts (35).The fact that insulin induces p125 Fak dephosphorylation suggests the existence of an antagonistic action of insulin on the integrin s...

Membrane-associated tyrosine phosphatase activities were studied in two distinct states of insulin resistance: diabetes and pregnancy. Using a novel immunoenzymatic assay with intact insulin-like growth factor-I (IGF-I) and insulin receptors as substrates, we show that phosphotyrosine-protein phosphatases (PTP-ases) from normal rat tissues induce a decrease in tyrosine phosphorylation of both receptors. Membrane fractions from kidney, brain, and liver contain the highest PTP-ase activity toward the insulin receptor. After 20-day streptozotocin-induced diabetes, PTP-ase activities are increased by 70% in the placenta, reduced by 40-50% in liver and skeletal muscle, and remained unchanged in the nonclassical insulin target tissues, kidney and brain. In general, the dephosphorylation of IGF-I receptor follows a pattern similar to that of insulin receptor except in red skeletal muscle in which it is not modified. Pregnancy also induces alterations of liver PTP-ases similar to those elicited by diabetes with a 50% reduction of insulin and IGF-I receptor dephosphorylation. This effect of pregnancy is further potentiated by diabetes. The alterations in the activity of hepatic PTP-ases from diabetic and pregnant rats are associated with a decreased autophosphorylation of the insulin receptor, suggesting that the diminution of phosphatase activity might be associated to the state of receptor phosphorylation and activation. Our data demonstrate that alterations of PTP-ases in insulin target tissues are found in two insulin-resistant states, one characterized by hyperinsulinemia, pregnancy and one by insulinopenia, streptozotocin-diabetes. These observations suggest a possible relationship between the defective activity of receptor tyrosine kinases and membrane-associated phosphatases from insulin responsive tissues.

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The effects of high glucose on insulin-receptor tyrosine kinase activity and gene expression were investigated in 3T3-HIR cells. Cells incubated for 48 h in the presence of 25 mM glucose showed a 5-fold increase in the amount of insulin receptors per cell, receptor autophosphorylation and phosphorylation of the exogenous substrate poly(Glu/Tyr) compared with cells grown in the absence of glucose but in the presence of 25 mM fructose. These effects were associated with a 4-fold stimulation in steady-state levels of insulin-receptor mRNA. Significant cellular glucose utilization and lactate production were observed in the presence of high glucose in the culture medium, indicating a functional glycolytic pathway in glucose-treated cells, but not in cells treated with fructose. Such a differential response to hexoses favours the hypothesis of a carbohydrate regulation via a glycolytic intermediate. This was further supported by a similar glucose-induced increase in mRNA levels of the enzyme glyceraldehyde-3-phosphate dehydrogenase. To test the hypothesis that the stimulatory effect of glucose on amount of insulin receptors and phosphorylation state could result from post-transcriptional modifications, cells exposed to glucose were incubated with actinomycin D, a potent inhibitor of gene transcription. In cells challenged with high glucose plus inhibitor, insulin-receptor mRNA half-life was increased from 1 to 3 h, indicating that posttranscriptional mechanisms are involved in these processes of glucose regulation. Inhibition of protein synthesis by cycloheximide induced an overexpression of insulin-receptor mRNA levels in the presence of glucose, suggesting that labile repressor protein(s) could be implicated in the effects of glucose. We conclude that (1) long-term culture with high glucose increases the amount of insulin receptors and their tyrosine kinase activity and (2) the glucose-induced increase in insulin-receptor mRNA levels can be accounted for, at least in part, by posttranscriptional events.

The activated insulin receptor phosphorylates docking proteins such as Src-Homology Collagen (Shc) and Insulin Receptor Substrate-1 (IRS-1), which then bind several proteins that contain a Src-Homology 2 (SH2) domain. Both Shc and IRS-1 associate with Growth Factor Receptor-Bound protein 2 (Grb2), an adaptor molecule. The hormone-receptor complex is then rapidly internalized through coated-pits. Dynamin, a 100 kDa protein with GTPase activity, is thought to play a crucial role in receptor-mediated endocytosis. In this study, we show that insulin induces tyrosine phosphorylation of dynamin in cells overexpressing human insulin receptors. Phosphorylation is observed rapidly, i.e. within 1 minute of insulin treatment. Moreover, exposure of cells to the hormone leads to co-immunoprecipitation of dynamin with Shc and with insulin receptor. Since dynamin constitutively associates with Grb2, it could be recruited to the insulin signaling complex through binding of Grb2 to tyrosine-phosphorylated Shc.