chico

EVOLUTIONARY HOMOLOGS part 2/3

Interaction of IRS proteins with PI 3-kinase

Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. It is proposed that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, it has been shown that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, the HSP could be separated into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. This IRS-1-PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells (Clark, 1998).

Phosphatidylinositol (PI) 3-kinase plays an important role in various insulin-stimulated biological responses, including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between PI 3-kinase and these biological responses is still unclear. Is targeting of the catalytic p110 subunit of PI 3-kinase to cellular membranes both sufficient and necessary to induce PI 3-kinase dependent signaling responses, as is characteristic of insulin action? Myc-tagged, membrane-targeted p110 [p110(CAAX)], and wild-type p110 [p110(WT)] in 3T3-L1 adipocytes were engineered by adenovirus-mediated gene transfer. Overexpressed p110(CAAX) exhibits approximately 2-fold increase in basal kinase activity in p110 immunoprecipitates; this further increases to approximately 4-fold with insulin. Even at this submaximal PI 3-kinase activity, p110(CAAX) fully stimulates p70 S6 kinase, Akt, 2-deoxyglucose uptake, and Ras, whereas, p110(WT) has little or no effect on these downstream effects. Interestingly, p110(CAAX) does not activate MAP kinase, despite its stimulation of p21(ras). Surprisingly, p110(CAAX) does not increase basal glycogen synthase activity, and inhibits insulin stimulated activity, indicative of cellular resistance to this action of insulin. p110(CAAX) also inhibits insulin stimulated, but not platelet-derived growth factor-stimulated mitogen-activated protein kinase phosphorylation, demonstrating that the p110(CAAX) induced inhibition of mitogen-activated protein kinase and insulin signaling is specific, and not due to some toxic or nonspecific effect on the cells. Moreover, p110(CAAX) stimulates IRS-1 Ser/Thr phosphorylation, and inhibits IRS-1 associated PI 3-kinase activity, without affecting insulin receptor tyrosine phosphorylation, suggesting that it may play an important role as a negative regulator for insulin signaling. In conclusion, these studies show that membrane-targeted PI 3-kinase can mimic a number of biologic effects normally induced by insulin. In addition, the persistent activation of PI 3-kinase induced by p110(CAAX) expression leads to desensitization of specific signaling pathways. Interestingly, the state of cellular insulin resistance is not global, in that some of insulin's actions are inhibited, whereas others are intact (Egawa, 1999).

IRS acts downstream of PDGF and Growth hormone receptors

Serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) has been implicated as a negative regulator of insulin signaling. Prior studies have indicated that this negative regulation by protein kinase C involves the mitogen-activated protein kinase and phosphorylation of serine 612 in IRS-1. In the present studies, the negative regulation by platelet-derived growth factor (PDGF) was compared with that induced by endothelin-1, an activator of protein kinase C. In contrast to endothelin-1, the inhibitory effects of PDGF does not require mitogen-activated protein kinase or the phosphorylation of serine 612. Instead, three other serines in the phosphorylation domain of IRS-1 (serines 632, 662, and 731) are required for the negative regulation by PDGF. In addition, the PDGF-activated serine/threonine kinase called Akt is found to inhibit insulin signaling. Moreover, this inhibition requires the same IRS-1 serine residues as the inhibition by PDGF. Finally, the negative regulatory effects of PDGF and Akt are inhibited by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), one of the downstream targets of Akt. These studies implicate the phosphatidylinositol 3-kinase/Akt kinase cascade as an additional negative regulatory pathway for the insulin signaling cascade (Li, 1999).

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. 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 is without a major effect. PDGF pretreatment for 60-90 min leads to a marked 80%-90% reduction in insulin stimulatable phosphotyrosine and IRS-1-associated PI3K activity. The functional consequences of this decrease in IRS-1-associated PI3K activity were examined. Interestingly, insulin stimulation of GLUT4 translocation and glucose transport is unaffected by 60-90 min of PDGF preincubation. Furthermore, insulin activation of Akt and p70(s6kinase: see Drosophila RPS6-p70-protein kinase), kinases downstream of PI3K, is unaffected by PDGF pretreatment. Wortmannin is 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), and of GLUT4 translocation and glucose transport (Staubs, 1998).

Insulin receptor substrate-1 (IRS-1) is tyrosine-phosphorylated in response to insulin resulting in association with and activation of phosphatidylinositol 3-kinase (PI 3-kinase), thereby initiating some of the effects of insulin. The insulin-like effects of growth hormone (GH) in adipocytes can be inhibited by the selective PI 3-kinase inhibitor wortmannin, suggesting a similar role for PI 3-kinase in GH action. IRS-1 is tyrosine-phosphorylated in a time- and dose-dependent manner in response to GH in primary rat adipocytes. This phosphorylation coincides with the extent of interaction between IRS-1 and the 85-kDa subunit of PI 3-kinase as evidenced by coimmunoprecipitation. Stimulation with 23 nM GH increases the PI 3-kinase activity associated with IRS1 4-fold. These data suggest that GH-induced tyrosine phosphorylation of IRS-1 and the subsequent docking of PI 3-kinase are important postreceptor events in GH action. The mechanism for the phosphorylation of IRS-1 induced by GH is unknown, but involvement of JAK2, the only known GH receptor-associated tyrosine kinase, seems possible (Ridderstrale, 1996).

Growth hormone (GH) and prolactin (PRL) binding to their receptors, which belong to the cytokine receptor superfamily, activate Janus kinase (JAK) 2 tyrosine kinase, thereby leading to their biological actions. GH mainly stimulates tyrosine phosphorylation of epidermal growth factor receptor and its association with Grb2, and concomitantly stimulates mitogen-activated protein kinase activity in liver, a major target tissue. Using specific antibodies, it has been shown that GH is also able to induce tyrosine phosphorylation of IRS-1/IRS-2 in liver. In addition, the major tyrosine-phosphorylated protein in anti-p85 phosphatidylinositol 3-kinase (PI3-kinase) immunoprecipitate from liver of wild-type mice is IRS-1, and IRS-2 in IRS-1 deficient mice, but not epidermal growth factor receptor. These data suggest that tyrosine phosphorylation of IRS-1 may be a major mechanism for GH-induced PI3-kinase activation in liver -- the physiological target organ of GH. PRL was able to induce tyrosine phosphorylation of both IRS-1 and IRS-2 in COS cells transiently transfected with PRLR and in CHO-PRLR cells. Moreover, tyrosine phosphorylation of IRS-3 is induced by both GH and PRL in COS cells transiently transfected with IRS-3 and their cognate receptors. By using the JAK2-deficient cell lines or by expressing a dominant negative JAK2 mutant, it has been shown that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3. Finally, a specific PI3-kinase inhibitor, wortmannin, completely blocks the anti-lipolytic effect of GH in 3T3 L1 adipocytes. Taken together, the role of IRS-1, -2, and -3 in GH and PRL signalings appears to be phosphorylated by JAK2, thereby providing docking sites for p85 PI3-kinase and activating PI3-kinase and its downstream biological effects (Yamauchi, 1998).

GH exerts a variety of metabolic and growth-promoting effects. GH induces activation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, JAK2, resulting in tyrosine phosphorylation of the GHR and activation of STAT (signal transducer and activator of transcription), Ras-mitogen-activated protein kinase, and phosphoinositol 3-kinase signaling pathways, among others. GH-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins has been demonstrated in vitro and in vivo. IRS-1 is a multiply phosphorylated cytoplasmic docking protein involved in metabolic and proliferative signaling by insulin, IL-4, and other cytokines, but the physiological role of IRS-1 in GH signaling is unknown. In murine 3T3-F442A pre-adipocytes, GH-dependent tyrosine phosphorylation of IRS-1 is observed as is specific GH-induced coimmunoprecipitation of IRS-1 with JAK2. This interaction was examined by in vitro affinity precipitation experiments with glutathione-S-transferase fusion proteins incorporating regions of rat IRS-1 and, as a source of JAK2, extracts of 3T3-F442A cells. Fusion proteins containing amino-terminal regions of IRS-1 that include the pleckstrin homology, phosphotyrosine-binding, and Shc and IRS-1 NPXY-binding domains, but not those containing other IRS-1 regions or glutathione-S-transferase alone, bind JAK2 from cell extracts. Tyrosine-phosphorylated JAK2 resulting from GH stimulation is included in the amino-terminal IRS-1 fusion precipitates; however, neither tyrosine phosphorylation of JAK2 nor treatment of cells with GH before extraction is necessary for the specific JAK2-IRS-1 interaction to be detected. In contrast, in this assay, specific insulin receptor association with the IRS-1 phosphotyrosine-binding, and Shc and IRS-1 NPXY-binding domains is insulin and phosphotyrosine dependent. To test for significance of IRS-1 with regard to GH signaling, IRS- and GHR-deficient 32D cells were stably reconstituted with the rabbit (r) GHR, either alone (32D-rGHR) or with IRS-1 (32D-rGHR-IRS-1). As assayed by three independent methods, GH induces proliferation in 32D-rGHR cells, even in the absence of transfected IRS-1. Notably, however, GH-induced proliferation is markedly enhanced in cells expressing IRS-1. Similarly, GH-induced mitogen-activated protein kinase activation is significantly augmented in IRS-1-expressing cells relative to that in cells harboring no IRS-1. These results indicate that IRS-1 enhances GH-induced proliferative signaling (Liang, 1999).

Biological effects of IRS proteins

continues...see chico: Evolutionary homologs part 3/3 | back to part 1/3

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