Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55 PIK , a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH 2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identity to those in p85. Phosphatidylinositol 3-kinase (PI-3 kinase) is a common signaling element which plays a role in the regulation of a broad array of biological responses by activated receptors for hormones, growth factors, cytokines, and antigens (6,13,15,42,46,48,61). It is composed of a 110-kDa catalytic subunit (p110) associated with an 85-kDa regulatory subunit (p85) that contains one Src homology 3 (SH3) domain, homology to the breakpoint cluster region (bcr) gene, two proline-rich motifs, and two SH2 domains (11). Interestingly, p110␣ displays dual catalytic specificity, as it phosphorylates the D-3 position of phosphatidylinositol and its phosphorylated derivatives and serine residues in p85 and insulin receptor substrate 1 (IRS-1) (12, 32). Mammalian p110 is homologous to VPS34, a Saccharomyces cerevisiae PI-3 kinase which is involved in vacuolar protein sorting (49); however, the molecular role of PI-3 kinase in mammalian cells is unclear (22).The p85 regulatory subunit has a broad potential to couple the PI-3 kinase to multiple signaling elements by employing its SH3 domain, proline-rich motifs, bcr homology region, or SH2 domains (24). Most activated receptors with tyrosine kinase activity engage the SH2 domains in p85 through phosphorylated YXXM motifs in the receptors themselves or a closely associated subunit (52). The platelet-derived growth factor receptor, one of the best-characterized systems, associates directly with the SH2 domains in p85 at a phosphorylated YMDM motif in the kinase insert region (61). Inhibition of PI-3 kinase catalytic activity with wortmannin or disruption of p85 function by site-directed mutagenesis blocks several growth factor-stimulated processes, including mitogenesis and antiapoptosis (61, 67), differentiation (27), receptor trafficking (23), chemotaxis (31, 45), membrane ruffling (29, 64), and insulin-stimulated glucose transport (7,16,44) and Xenopus oocyte maturation (8, 9). In addition, PI-3 kinase appears to be required for the stimulation of p70S6k by platelet-derived growth factor and insulin and probably other growth factors (7,10,40). On the basis of these results, PI-3 kinase plays a central role in cellular signaling.Insulin regulates PI-3 kinase by tyrosine phosphorylation of IRS-1 and IRS-2, multipotential docking proteins which contain multiple potential tyrosine phosphorylation sites, including several YXXM motifs (41, 58). In addition to insulin and insulin-like growth factor 1 (IGF-1), ...

GRB-2 is a small S112-and SH3 domain-containing adapter protein that associates with the mammalian SOS homolog to regulate p2l1 during growth factor signaling. During insulin stimulation, GRB-2 binds to the phosphorylated Y895VNI motif of IRS-1. Substitution of Tyr-895 with phenylalanine (IRS-1F-895) prevented the IRS-1-GRB-2 association in vivo and in vitro. The myeloid progenitor cell line, 32-D, is insensitive to insulin because it contains few insulin receptors and no IRS-i. Coexpression of IRS-1 or IRS-1F-s95 with the insulin receptor was required for insulin-stimulated mitogenesis in 32-D cells, while expression of the insulin receptor alone was sufficient to mediate insulin-stimulated tyrosine phosphorylation of Shc and activation of p2l1 and mitogen-activated protein (MAP) kinase. The Shc-GRB-2 complex formed during insulin stimulation is a possible mediator of p21r' and MAP kinase activation in IRS-1-deficient 32-D cells. Interestingly, IRS-1, but not IRS-1F-s95, enhanced the stimulation of MAP kinase by insulin in 32-D cells expressing insulin receptors. Thus, IRS-1 contributes to the stimulation of MAP kinase by insulin, probably through formation of the IRS-1-GRB-2 complex at Tyr-895. Our results suggest that the Shc-GRB-2 complex and the activation of p21r'-dependent signaling pathways, including MAP kinase, are insufficient for insulin-stimulated mitogenesis and that the essential function(s) of IRS-1 in proliferative signaling is largely unrelated to IRS-1-GRB-2 complex formation.IRS-1 is a 131-kDa protein that undergoes tyrosine phosphorylation during stimulation of cells with insulin, insulin-like growth factor 1 (IGF-1), and interleukin 4 (IL-4) and immediately associates with signaling molecules which contain Src homology 2 domains (SH2 proteins) (23,25,36,37,40,41). Overexpression of IRS-1 increases the proliferative response of Chinese hamster ovary cells to insulin and IGF-1 (36), whereas reduction of the IRS-1 level reduces the response (42). Moreover, 32-D myeloid progenitor cells are insensitive to insulin, IGF-1, and IL-4 because they lack IRS-1 or a functionally related molecule called 4PS (41). Expression of IRS-1 in 32-D cells rescues the mitogenic response to IGF-1 and IL-4, whereas coexpression of IRS-1 and insulin receptors (IRs) rescues the insulin response (40,41). Thus, IRS-1 appears to play an essential role for some insulin responses by linking the receptor tyrosine kinase to key SH2 proteins.IRS-1 contains more than 20 potential tyrosine phosphorylation sites, several of which bind specifically to the SH2 domains in various signaling proteins, including phosphatidylinositol (Ptdlns) 3'-kinase, GRB-2, Syp, and nck (17,18,33,37). The SH2 domains in Ptdlns 3'-kinase preferentially bind to phosphopeptides containing Y-460, Y-608, Y-939, and Y-987, whereas GRB-2 and SH-PTP2 bind phosphopeptides containing Y-895 and Y-1172, respectively (35). Tyrosine phosphorylation of IRS-1 acts as the switch to control these interactions, whereas the surrounding amino acid sequences select the ...

; IRS-1 YCT was tyrosine phosphorylated but also failed to mediate these signaling events. Neither IRS-1 3YMXM nor IRS-1 YCT mediated activation of mitogen-activated protein kinases. IRS-1 F18 and IRS-1 YCT partially mediated similar levels of insulin-stimulated mitogenesis at high insulin concentrations, however, suggesting that IRS-1 contains phosphotyrosine-independent elements which effect mitogenic signals, and that the sites in IRS-1 YCT do not augment this signal. IRS-1 3YMXM mediated the maximal mitogenic response to insulin, although the response to insulin was more sensitive with wild-type IRS-1. By contrast, the association of IRS-1 3YMXM with PI 3-kinase was more sensitive to insulin than the association with IRS-1. Thus, the binding of SH2 proteins (such as PI 3-kinase) by YMXM motifs in IRS-1 is an important element in the mitogenic response, but other elements are essential for full mitogenic sensitivity.The IRS proteins (IRS-1 and IRS-2) are endogenous cellular substrates of the insulin and insulin-like growth factor 1 (IGF-1) receptor tyrosine kinases, as well as substrates for various cytokine receptors which activate various members of the Janus kinase family (22,41,42,47,48). Tyrosine phosphorylation plays a central role in the mechanism of IRS protein signaling by providing docking sites for proteins containing isoforms of the Src homology 2 (SH2) domain (25,38,39). Since the phosphorylation sites in IRS-1 are situated in various amino acid sequence motifs, IRS-1 engages numerous SH2 proteins, including p85, Grb-2, Fyn, Nck, and SHP2 (SHPTP2, Syp, and PTP1D) (10,15,36). IRS proteins contain several phosphorylation sites in YXXM motifs, which are selective binding sites for the SH2 domains in the regulatory subunits (p85 and p55 PIK ) of phosphatidylinositol (PI) 3Ј-kinase (22,28,41). During insulin stimulation, IRS proteins mediate the direct activation of PI 3Ј-kinase, which appears to play essential roles in the activation of the p70 s6k and the akt kinase (21, 22) and the serine phosphorylation of eIF4e and PHAS-I as well as in mediating various biological responses including vesicle movement (including glucose transporter translocation), mitogenesis, general protein synthesis, receptor endocytosis, and chemotaxis, among other things (22,25,48).While the plethora of signaling events apparently mediated by PI 3Ј-kinase raises questions about whether PI 3Ј-kinase is a regulator of specific cellular events or whether it is a global potentiator of cellular signaling, it is obviously an important molecule in the transmission of downstream signals by tyrosine kinases. The 110-kDa PI 3Ј-kinases are coupled to tyrosine kinases by regulatory adapter molecules (p85 and p55 PIK ) which use their SH2 domains to bind tyrosyl-phosphorylated YMXM motifs (14, 28). The 110-kDa catalytic domain (p110) is activated by the binding of these tyrosyl-phosphorylated YMXM motifs (2). In addition to its ability to phosphorylate PI, p110 also phosphorylates certain proteins on serine residues (3,8,14,17). The SH2 d...