Since their discovery, protein tyrosine phosphatases have been speculated to play a role in tumor suppression because of their ability to antagonize the growthpromoting protein tyrosine kinases. Recently, a tumor suppressor from human chromosome 10q23, called PTEN or MMAC1, has been identified that shares homology with the protein tyrosine phosphatase family. Germ-line mutations in PTEN give rise to several related neoplastic disorders, including Cowden disease. A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. Here we report that a missense mutation in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or substrate-trapping forms of PTEN in HEK293 cells altered the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the phosphatase in PTEN-deficient tumor cell lines resulted in the inhibition of protein kinase (PK) B͞Akt and regulation of cell survival.

PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1(-/-) cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase.

. dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1.

Insulin stimulated protein kinase Ba (PKBa) more than 10-fold and decreased glycogen synthase kinase-3 (GSK3) activity by 50 ± 10% in skeletal muscle and adipocytes. Rapamycin did not prevent the activation of PKB, inhibition of GSK3 or stimulation of glycogen synthase up to 5 min. Thus rapamycin-insensitive pathways mediate the acute effect of insulin on glycogen synthase in the major insulin-responsive tissues. The small and very transient effects of EGF on phosphatidylinositol (3,4,5)P 3 PKBa and GSK3 in adipocytes, compared to the strong and sustained effects of insulin, explains why EGF does not stimulate glucose uptake or glycogen synthesis in adipocytes.

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The mechanism by which leptin increases ATP-sensitive K ؉ (K ATP ) channel activity was investigated using the insulin-secreting cell line, CRI-G1. Wortmannin and LY 294002, inhibitors of phosphoinositide 3-kinase (PI3-kinase), prevented activation of K ATP channels by leptin. The inositol phospholipids phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate (PtdIns(3,4,5)P 3 ) mimicked the effect of leptin by increasing K ATP channel activity in whole-cell and insideout current recordings. LY 294002 prevented phosphatidylinositol bisphosphate, but not PtdIns(3,4,5)P 3 , from increasing K ATP channel activity, consistent with the latter lipid acting as a membrane-associated messenger linking leptin receptor activation and K ATP channels. Signaling cascades, activated downstream from PI 3-kinase, utilizing PtdIns(3,4,5)P 3 as a second messenger and commonly associated with insulin and cytokine action (MAPK, p70 ribosomal protein-S6 kinase, stress-activated protein kinase 2, p38 MAPK, and protein kinase B), do not appear to be involved in leptin-mediated activation of K ATP channels in this cell line. Although PtdIns(3,4,5)P 3 appears a plausible and attractive candidate for the messenger that couples K ATP channels to leptin receptor activation, direct measurement of PtdIns(3,4,5)P 3 demonstrated that insulin, but not leptin, increased global cellular levels of PtdIns(3,4,5)P 3 . Possible mechanisms to explain the involvement of PI 3-kinases in K ATP channel regulation are discussed.The hormone leptin, secreted by adipocytes, has a major influence on body weight homeostasis (1, 2). Although the hypothalamus is considered the main target for leptin, particularly with respect to body weight regulation, it is clear that this hormone has distinct actions on other peripheral, target organs. There have been several reports that leptin reduces insulin secretion from pancreatic beta cells (3-6), although this view is not shared by all investigators (7). One mechanism proposed to explain the leptin-induced reduction in insulin secretion is via activation of ATP-sensitive K ϩ (K ATP ) channels (8, 9). This increase in potassium current results in beta cell hyperpolarization, reduced calcium entry, and hence decreased insulin secretion. In addition, there are features common to both insulin-secreting cells and leptin-sensitive hypothalamic neurones (10, 11), most notably glucose responsiveness and the presence of K ATP channels, which are activated by exposure of the cells to leptin. The apparent involvement of both leptin receptors and K ATP channel activation in key systems involved in metabolic homeostasis has led us to examine the likely signal transduction pathways underlying this effect.The leptin receptor belongs to the class I cytokine receptor superfamily (1, 2), members of which are thought to signal via janus-tyrosine kinases. Activated janus-tyrosine kinases can mediate signaling via insulin receptor substrate proteins (12-14), which following tyrosine phosphorylation become docking sites for Sr...

The micro‐organism Dictyostelium uses extracellular cAMP to induce chemotaxis and cell differentiation. Signals are transduced via surface receptors, which activate G proteins, to effector enzymes. The deduced protein sequence of Dictyostelium discoideum phosphatidylinositol‐specific phospholipase C (PLC) shows strong homology with the mammalian PLC‐delta isoforms. To study the role of PLC in Dictyostelium, a plc‐ mutant was constructed by disruption of the PLC gene. No basal or stimulated PLC activity could be measured during the whole developmental programme of the plc‐ cells. Loss of PLC activity did not result in a visible alteration of growth or development. Further analysis showed that developmental gene regulation, cAMP‐mediated chemotaxis and activation of guanylyl and adenylyl cyclase were normal. Although the cells lack PLC activity, inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] was present at only slightly lower concentrations compared with control cells. Mass analysis of inositol phosphates demonstrated the presence of a broad spectrum of inositol phosphates in Dictyostelium, which was unaltered in the plc‐ mutant. Cell labelling experiments with [3H]inositol indicated that [3H]Ins(1,4,5)P3 was formed in a different manner in the mutant than in control cells.

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...