Signaling by phosphatidylinositol (PI) 3-kinases is mediated by 3-phosphoinositides, which bind to Pleckstrin homology (PH) domains that are present in a wide spectrum of proteins. PH domains can be classified into three groups based on their different lipid binding specificities. Distinct 3-phosphoinositides can accumulate upon PI 3-kinase activation in cells in response to different stimuli and mediate specific cellular responses. In Swiss 3T3 mouse fibroblasts, oxidative stress induced by 1 mM H 2 O 2 caused almost exclusive accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ), whereas osmotic stress increased both phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) and PtdIns-(3,4)P 2 levels. The increase in PtdIns(3,4)P 2 levels, caused by oxidative stress, correlated with the activation of protein kinase B, which has a promiscuous PH domain that binds both PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 . p70 S6 kinase, another signaling component downstream of PI 3-kinase, however, was not activated by this oxidative stress-induced increase in PtdIns(3,4)P 2 levels. Increased PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 levels in response to osmotic stress did not correlate with protein kinase B activation, because of concomitant activation of an inhibitory pathway, but p70 S6 kinase was activated by osmotic stress. These results demonstrate that PtdIns(3,4)P 2 can accumulate independently of PtdIns(3,4,5)P 3 and exerts a pattern of cellular responses that is distinct from that induced by accumulation of PtdIns(3,4,5)P 3 .The significance of PI 1 3-kinases in the regulation of a wide spectrum of cellular signaling events has been well established (1, 2). Based on structural features and substrate specificity, three classes of catalytic subunits of PI 3-kinases have so far been recognized (2, 3). The Type I class, members of which phosphorylate PtdIns, PtdIns4P, and PtdIns(4,5)P 2 in vitro but utilize PtdIns(4,5)P 2 as the likely substrate in vivo, is subdivided into Type I a and Type 1 b . Type I a PI 3-kinases interact with adaptor proteins containing SH2 domains that bind phosphotyrosine residues, linking this class to tyrosine kinase signaling cascades. Type 1 b PI 3-kinases are stimulated by Gprotein ␥ subunits and do not interact with SH2 domain containing adaptor proteins but with p101, a novel adaptor protein that has no homology with known proteins. Type II PI 3-kinases phosphorylate PtdIns and PtdIns4P in vitro (and not PtdIns(4,5)P 2 ) and contain a C2 domain, implicated in lipid binding. Type III PI 3-kinases only phosphorylate PtdIns and are thought to be constitutively active. They can be recruited and regulated by their adaptor, a dual specificity protein kinase, with which they form heterodimers.The principle enzymatic activity of PI 3-kinases involves the phosphorylation of phosphoinositides on the 3-position of the inositol ring, resulting in the formation of 3-phosphoinositides, which can either directly interact with appropriate modules of specific target proteins or first under...