IRS-4 mediates protein kinase B signaling during insulin stimulation without promoting antiapoptosis.

Insulin receptor substrate (IRS) proteins play a central role in signal transduction by the receptors for insulin, insulin-like growth factor 1 (IGF-1), and a growing number of cytokines and integrins (19, 34, 58). IRS proteins are composed of an NH2-terminal pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, followed by a COOH-terminal tail containing multiple tyrosine phosphorylation motifs (48). The PH and PTB domains are well conserved in each IRS protein and have a common tertiary structure but function differently to couple IRS proteins to the activated insulin receptor (10, 26, 57). PH domains are thought to bind membrane phospholipids or acidic motifs of various proteins, whereas the PTB domain interacts with the phosphorylated NPEY motif in the juxtamembrane region of the insulin receptor β-subunit; both interactions promote tyrosine phosphorylation of multiple tyrosine residues in the COOH terminus. The COOH terminus is poorly conserved among the IRS proteins; in IRS-1 (the first cloned and best-characterized IRS protein) it contains 18 potential tyrosine phosphorylation sites in amino acid sequence motifs that directly bind to Src homology-2 domains in several proteins, including phosphatidylinositol (PI) 3-kinase regulatory subunits (p85α, p55α p50α, p85β, and p55PIK), Grb-2, Nck, c-fyn, and SHP-2 (58). The other IRS proteins contain common and unique phosphorylation motifs, suggesting that they mediate overlapping and distinct biological signals. One region of predicted overlap in the signals mediated by IRS-1, IRS-2, and IRS-4 is the binding and activation of PI 3-kinase; a conspicuous example of dissimilarity is the absence of SHP-2 binding motifs in IRS-4. PI 3-kinase is a heterodimeric signaling enzyme composed of a catalytic subunit (p110) associated with an SH2 domain-containing regulatory subunit (e.g., p55PIK, p85α, and p85β) that binds to tyrosine-phosphorylated YXXM motifs on signaling proteins. Since all of the IRS proteins contain numerous YXXM motifs, they are all expected to bind and activate p85-associated PI 3-kinase. The lipid products of PI 3-kinase mediate the activation of several serine/threonine kinases, including PDK1/PDK2, protein kinase B (PKB)/Akt, PKCζ and PKCλ, p70s6k, and others (3, 29, 32, 52, 53). These serine/threonine kinases play various roles in the regulation of the many biological responses linked to the activation of PI 3-kinase, including activation of glucose transport and glycogen synthesis, protein synthesis, lipogenesis and antilipolysis, and mitogenesis and antiapoptosis (12, 14, 16, 22, 38). In vivo and in vitro experiments reveal important differences in the signaling capacity of IRS-1 and IRS-2. IRS-1 and IRS-2 display differential sensitivities for binding various SH2 proteins (46). Moreover, IRS-2 expressed in fibroblasts lacking IRS-1 does not reconstitute normal insulin–IGF-1 signaling (9). Mice lacking IRS-1 are small and mildly insulin resistant but never develop diabetes (5). In contrast, mice lacking IRS-2 are insulin resistant with abnormal glucose tolerance at birth and progressively develop fasting hyperglycemia with reduced β-cell mass (54; D. J. Withers, D. J. Burks, H. H. Towery, S. L. Altamuro, C. L. Flint, and M. F. White, unpublished data). Together, these results suggest that IRS-2 is critical for normal carbohydrate metabolism and that IRS-1 is important for somatic cell growth (55). Previous results suggest that IRS-4 has similar signaling potential to IRS-1 and IRS-2; however, our results with 32DIR cells revealed that IRS-4 activates PKB/Akt and promotes BAD phosphorylation without the activation of p70s6k or the inhibition of apoptosis. These differences might have important consequences for the specific tissues that express IRS-4.