IRS-proteins couple insulin and IGF receptors to various signal pathways, including the PI 3-kinase ^ protein kinase B (PKB)/AKT cascade and the Grb2/SOS ^p21ras cascade (Fig. 2). Activation of class 1A PI 3-kinase is required for many insulin responses, including the stimulation of glucose uptake, glycogen synthesis, and gene transcription . PI 3-kinase is composed of a catalytic and a regulatory subunit. Catalytic subunits encoded by Pik3ca (p110a), Pik3cb (p110P), and Pik3cd (p1105) associate noncovalently with a regulatory subunit encoded by Pik3r2 (p85P), Pik3r3 (p55PIK), or the alternatively spliced Pik3r1 (p85a, p55a, and p50a) . Pik3r1 and Pik3r2 are ubiquitously expressed; p85a is usually more abundant than p85p, whereas Pik3r3 displays a restricted pattern of expression . Each regulatory subunit contains a p110-binding region flanked by an SH2 domain, and p85a and p85p have an
NH2-terminal SH3-domain that is replaced by short unique sequences in p55a, p50a, and p55PIK. The importance of these structural differences is unknown.
During insulin stimulation, phosphorylated YMXM motifs in IRS-proteins occupy both SH2 domains in the regulatory subunits which directly activates the PI 3-kinase . Products of the PI 3-kinase, including phosphatidyli-nositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, recruit and activate the PDK(1/2) ^ PKB (1/2/3) cascade (Fig. 2). During colocalization at the plasma membrane, PDKs phosphorylate and activate PKB isoforms, which control various biological processes, including glucose transport, protein synthesis, glycogen synthesis, cell proliferation, and cell survival in various cells and tissues (Fig. 2) [14,50,51].
The role of IRS-proteins in PI 3-kinase signaling is complex, as IRS1 and IRS2 play distinct regulatory roles in liver and muscle. Basal PI 3-kinase activity in muscle and liver is
Figure 2 IRS-proteins coordinate downstream signaling pathways; the activation of intracellular signaling pathways by insulin is shown. The diversity of insulin action in various tissues is partly explained by the different signaling pathways activated by the hormone. The two main limbs that propagate the signal generated through the insulin receptor are the insulin receptor substrate/phosphatidylinositol 3-kinase (IRS/PI3K) pathway, and the Ras/mitogen-activated protein kinase (MAPK) pathway. Activation of the receptors for insulin and IGF1 results in tyrosine phosphorylation of the IRS-proteins. The IRS-proteins bind PI 3-kinase, Grb2/SOS, and SHP2. The GRb2/SOS complex mediates the activation of p21ras, thereby activating the ras ^ raf ^ MEK ^ MAP kinase cascade. SHP2 feeds back to inhibit IRS-protein phos-phorylation by direct dephosphorylation of the IRS-protein but might also transmit an independent signal to activate MAP kinase. The activated MAP kinase phosphorylates p90rsk, which itself phosphorylates c-fos, thus increasing its transcriptional activity. MAP kinase also phosphorylates ELK1, also increasing its transcriptional activity. The activation of PI 3-kinase by IRS-protein recruitment results in the generation of PI3,4P2 and PI3,4,5P3 (antagonized by the action of PTEN or SHIP2). In aggregate, PI3,4P2 and PI3,4,5P3 activate a variety of downstream signaling kinases, including mTOR, which regulates protein synthesis via PHAS/p70s6k/EIF4. These lipids also activate alternate PKC iso-forms and PDK isoforms. The PDKs activate protein kinase B (PKB), which appears to mediate glucose transport in concert with the atypical PKC isoforms. PKB also regulates GSK3, which may regulate glycogen synthesis, and a variety of regulators of cell survival. PKB-mediated BAD phosphorylation inhibits apoptosis, and phosphorylation of the forkhead proteins results in their sequestration in the cytoplasm, in effect inhibiting their transcriptional activity. Abbreviations: AKT, product of the akt protooncogene; GAP, guanosine-triphosphatase-associated protein; GLUT4, glucose transporter 4; GRB-2, factor receptor binding protein 2; GSK3, glycogen synthase kinase 3; MAPKK, MAPK kinase; PDK, PI-dependent protein kinase; PKC, protein kinase C; SOS, son-of-sevenless.
elevated upon disruption of IRS2, which reduces the relative increase during insulin stimulation (Fig. 3). By contrast, without IRS1, basal PI 3-kinase activity is reduced significantly while insulin-stimulated activity is preserved, so the relative activation by insulin is increased. Apparently, IRS1 has a greater impact on basal PI 3-kinase activity and IRS2 has it greatest impact on insulin-stimulated activity. Thus, IRS2 might be the principle regulator of the metabolic insulin actions in liver and muscle .
The PI 3-kinase regulatory subunits also influence the signal specificity and signal strength. Complete disruption of the Pik3r1 gene that eliminates p85a, p55a, and p50a causes death of newborn mice . By contrast, complete disruption of Pik3r2 is not lethal . Interestingly, mice lacking p850 display improvised insulin sensitivity. These results suggest that the strength of the IRS ^ PI 3-kinase signal might be determined, at least in part, by the stoichiom-etry between the regulatory and catalytic subunits. Because the number of regulatory subunits generally exceeds that of the catalytic subunits, a competition apparently exists for IRS-protein binding sites between catalytically competent heterodimers and catalytically deficient regulatory monomers [48,54]. This hypothesis is supported by the finding that the selective reduction of p85a increases insulin sensitivity . Although the stoichiometry between IRS-proteins and PI 3-kinase regulatory subunits is important for signal strength, other mechanisms might also contribute to feedback inhibition through direct inhibition of IRS-protein function.
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