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.

The activation of phosphoinositide 3-kinase (PI3K) by insulin represents a key signalling event in the hormonal stimulation of diverse cellular responses including glucose transport and glycogen synthesis. The activation of PI3K increases the production of 4,5 trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol 3,4 bisphosphate [PtdIns(3,4)P 2 ]), which act as important signalling intermediates in the downstream activation of the serine/threonine kinase, Protein Kinase B (PKB/Akt). Activation of PKB depends upon its phosphorylation on two key amino acid residues, Thr 308 and Ser 473, with full activation requiring the phosphorylation of both [1]. The Nterminal domain of PKB contains a pleckstrin homology (PH) domain, which is thought to be critical in allowing the kinase to interact with 3-phospho- Diabetologia (2001) Abstract Aims/hypothesis. Increased cellular production of ceramide has been implicated in the pathogenesis of insulin resistance and in the impaired utilisation of glucose. In this study we have used L6 muscle cells to investigate the mechanism by which the short-chain ceramide analogue, C 2 -ceramide, promotes a loss in insulin sensitivity leading to a reduction in insulin stimulated glucose transport and glycogen synthesis. Method. L6 muscle cells were pre-incubated with C 2 -ceramide and the effects of insulin on glucose transport, glycogen synthesis and the activities of key molecules involved in proximal insulin signalling determined.Results. Incubation of L6 muscle cells with ceramide (100 mmol/l) for 2 h led to a complete loss of insulinstimulated glucose transport and glycogen synthesis. This inhibition was not due to impaired insulin receptor substrate 1 phosphorylation or a loss in phosphoinositide 3-kinase activation but was caused by a failure to activate protein kinase B. This defect could not be attributed to inhibition of 3-phosphoinositidedependent kinase-1, or to impaired binding of phosphatidylinositol 3,4,5 triphosphate (PtdIns(3,4,5)P 3 ) to the PH domain of protein kinase B, but results from the inability to recruit protein kinase B to the plasma membrane. Expression of a membrane-targetted protein kinase B led to its constitutive activation and an increase in glucose transport that was not inhibited by ceramide. Conclusions/interpretation. These findings suggest that a defect in protein kinase B recruitment underpins the ceramide-induced loss in insulin sensitivity of key cell responses such as glucose transport and glycogen synthesis in L6 cells. They also suggest that a stimulated rise in PtdIns(3,4,5)P 3 is necessary but not sufficient for protein kinase B activation in this system. [Diabetologia (2001) 44: 173±183]

The PTEN tumour suppressor is a lipid and protein phosphatase that inhibits phosphoinositide 3-kinase (PI3K)-dependent signalling by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate (PtdInsP 3 ). Here, we discuss the concept of PTEN as an 'interfacial enzyme', which exists in a high activity state when bound transiently at membrane surfaces containing its substrate and other acidic lipids, such as PtdIns(4,5)P 2 and phosphatidylserine (PtdSer). This mechanism ensures that PTEN functions in a spatially restricted manner, and may explain its involvement in forming the gradients of PtdInsP 3 , which are necessary for generating and/or sustaining cell polarity during motility, in developing neurons and in epithelial tissues. Coordinating PTEN activity with alternative mechanisms of PtdInsP 3 metabolism, by the tightly regulated SHIP 5-phoshatases, synthesizing the independent second messenger PtdIns(3,4)P 2 , may also be important for cellular polarization in some cell types. Superimposed on this interfacial mechanism are additional post-translational regulatory processes, which generally act to reduce PTEN activity. Oxidation of the active site cysteine residue by reactive oxygen species and phosphorylation of serine/threonine residues at sites in the C-terminus of the protein inhibit PTEN. These phosphorylation sites also appear to play a role in regulating both stability and localization of PTEN, as does ubiquitination of PTEN. Because genetic studies in mice show that the level of expression of PTEN in an organism profoundly influences tumour susceptibility, factors that regulate PTEN, localization, activity and turnover should be important in understanding its biological functions as a tumour suppressor.

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The aggregation of human platelets is an important physiological hemostatic event contingent upon receptordependent activation of the surface integrin ␣ IIb ␤ 3 and subsequent binding of fibrinogen. Aggregating platelets form phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ), which has been reported to stimulate in vitro the activity of the proto-oncogenic protein kinase PKB/Akt, as has phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ). It has been assumed that PtdIns(3,4)P 2 is synthesized by either 5-phosphatase-catalyzed hydrolysis of PtdIns(3,4,5)P 3 produced by phosphoinositide 3-kinase (PI3K) or phosphorylation by PI3K of PtdIns4P. We investigated the route(s) by which PtdIns(3,4)P 2 is formed after directly activating ␣ IIb ␤ 3 with anti-ligand-induced binding site Fab fragment and report that aggregation does not lead to the generation of PtdIns(3,4,5)P 3 , but to transient formation of PtdIns3P and generation of PtdIns(3,4)P 2 , the latter primarily by PtdIns3P 4-kinase. Both this novel pathway and the activation of PKB/Akt are inhibited by the PI3K inhibitor, wortmannin, and the calpain inhibitor, calpeptin, constituting the first evidence that PtdIns(3,4)P 2 can stimulate PKB/Akt in vivo in the absence of PtdIns(3,4,5)P 3 . Integrin-activated generation of the second messenger PtdIns(3,4)P 2 thus depends upon a route distinct from that known to be utilized initially by growth factors. This pathway is of potential general relevance to the function of integrins.Human platelets have provided a model system for a variety of signal transduction events, including integrin-based signaling. Platelets can be activated by agents that include agonists for the thrombin receptor (THR-R), 1 leading to a change in integrin ␣ IIb ␤ 3 conformation to one that binds plasma fibrinogen (FIB) and results in aggregation. The change in integrin is dependent partially upon the activation of an 85 K D subunit-containing form of PI3K (1, 2), which acts in vivo on PtdIns(4,5)P 2 and rapidly generates PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 , but not PtdIns3P (3, 4). Late (post-aggregation) accumulations of PtdIns(3,4)P 2 , but not the levels of PtdIns(3,4,5)P 3 (5), have been found to be regulated by extracellular Ca 2ϩ and binding of FIB to ␣ IIb ␤ 3 (5, 6). Other work has shown that THR-R-dependent accumulation of PtdIns(3,4)P 2 can be impaired by calpeptin, an inhibitor of the Ca 2ϩ -dependent protease calpain, which is activated under these conditions (7-9). Norris et al. (9) have suggested that calpain hydrolytically inactivates PtdIns(3,4)P 2 4-phosphatase, thereby elevating PtdIns(3,4)P 2 . The rise in PtdIns(3,4)P 2 that follows THR-R stimulation has been correlated kinetically with the regulation of the serine-threonine kinase PKB/Akt (10), although a role for the earlier elevation in PtdIns(3,4,5)P 3 levels could not be discounted by these studies. Indeed, both PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 are potent stimuli for PDK1, which phosphorylates PKB/Akt and thereby activates it (11). Another report has a...

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour-suppressor protein is a phosphoinositide 3-phosphatase which antagonizes phosphoinositide 3-kinase-dependent signalling by dephosphorylating PtdIns(3,4,5)P3. Most tumour-derived point mutations of PTEN induce a loss of function, which correlates with profoundly reduced catalytic activity. However, here we characterize a point mutation at the N-terminus of PTEN, K13E from a human glioblastoma, which displayed wild-type activity when assayed in vitro. This mutation occurs within a conserved polybasic motif, a putative PtdIns(4,5)P2-binding site that may participate in membrane targeting of PTEN. We found that catalytic activity against lipid substrates and vesicle binding of wild-type PTEN, but not of PTEN K13E, were greatly stimulated by anionic lipids, especially PtdIns(4,5)P2. The K13E mutation also greatly reduces the efficiency with which anionic lipids inhibit PTEN activity against soluble substrates, supporting the hypothesis that non-catalytic membrane binding orientates the active site to favour lipid substrates. Significantly, in contrast to the wild-type enzyme, PTEN K13E failed either to prevent protein kinase B/Akt phosphorylation, or inhibit cell proliferation when expressed in PTEN-null U87MG cells. The cellular functioning of K13E PTEN was recovered by targeting to the plasma membrane through inclusion of a myristoylation site. Our results establish a requirement for the conserved N-terminal motif of PTEN for correct membrane orientation, cellular activity and tumour-suppressor function.

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Stimulation of serum-starved human embryonic kidney (HEK) 293 cells with either the phorbol ester, 12-Otetradecanoylphorbol-13-acetate (TPA), or insulin resulted in increases in the phosphorylation of 4E-BP1 and p70 S6 kinase, eIF4F assembly, and protein synthesis. All these effects were blocked by rapamycin, a specific inhibitor of mTOR. Phosphatidylinositol 3-kinase and protein kinase B were activated by insulin but not by TPA. Therefore TPA can induce eIF4F assembly, protein synthesis, and the phosphorylation of p70 S6 kinase and 4E-BP1 independently of both phosphatidylinositol 3-kinase and protein kinase B. Using two structurally unrelated inhibitors of MEK (PD098059 and U0126), we provide evidence that Erk activation is important in TPA stimulation of eIF4F assembly and the phosphorylation of p70 S6 kinase and 4E-BP1 and that basal MEK activity is important for basal, insulin, and TPA-stimulated protein synthesis. Transient transfection of constitutively active mitogen-activated protein kinase interacting kinase 1 (the eIF4E kinase) indicated that inhibition of protein synthesis and eIF4F assembly by PD098059 is not through inhibition of eIF4E phosphorylation but of other signals emanating from MEK. This report also provides evidence that increased eIF4E phosphorylation alone does not affect the assembly of the eIF4F complex or general protein synthesis.Protein synthesis is regulated by a variety of stimuli including insulin (1) and phorbol esters (2-6). A key step in the regulation of protein synthesis is the assembly of the initiation complex eIF4F, 1 containing the initiation factors eIF4G, a large scaffolding protein, eIF4E, the protein which binds to the 5Ј cap structure (7-methylguanosine triphosphate) and eIF4A, a bidirectional RNA helicase (reviewed by Sonenberg (7)). The formation of the eIF4F complex (usually defined operationally as the association of eIF4G with eIF4E) is regulated by a variety of stimuli through multiple phosphorylation events. Two principal mechanisms have been suggested to play an important role in regulating eIF4F assembly. First, it has been proposed that the phosphorylation of eIF4E may be important in the formation of the eIF4F complex, as eIF4E associated with eIF4G is more highly phosphorylated than free eIF4E (8, 9) and since increased phosphorylation of eIF4E often parallels increases in total protein synthesis (reviewed by Kleijn et al. (10)). Recent data have provided evidence that eIF4E is phosphorylated by MAP kinase interacting kinase 1 (Mnk1), which is itself phosphorylated by both p38-MAPK and Erk (11-15). The second mechanism regulating eIF4F assembly involves a family of eIF4E-binding proteins (4E-BPs, archetype 4E-BP1) whose affinity for eIF4E is regulated through their phosphorylation (16 -20). 4E-BP1 binds to a site on eIF4E which overlaps the binding site for eIF4G (18,21,22). 4E-BP1 thus competes with eIF4G for binding to eIF4E and can therefore inhibit eIF4F complex formation. Phosphorylation of 4E-BP1 leads to a reduction in its affinity for eIF4E and...