Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN.

PTEN is a tumour suppressor with phosphatase activity in vitro against both lipids and proteins and other potential non-enzymatic mechanisms of action. Although the importance of PTEN's lipid phosphatase activity in regulating the PI3K signalling pathway is recognized, the significance of PTEN's other mechanisms of action is currently unclear. In this study, we describe the systematic identification of a PTEN mutant, PTEN Y138L, with activity against lipid, but not soluble substrates. Using this mutant, we provide evidence for the interfacial activation of PTEN against lipid substrates. We also show that when re-expressed at physiological levels in PTEN null U87MG glioblastoma cells, the protein phosphatase activity of PTEN is not required to regulate cellular PtdInsP(3) levels or the downstream protein kinase Akt/PKB. Finally, in three-dimensional Matrigel cultures of U87MG cells similarly re-expressing PTEN mutants, both the protein and lipid phosphatase activities were required to inhibit invasion, but either activity alone significantly inhibited proliferation, albeit only weakly for the protein phosphatase activity. Our data provide a novel tool to address the significance of PTEN's separable lipid and protein phosphatase activities and suggest that both activities suppress proliferation and together suppress invasion.

Understanding PTEN regulation: PIP2, polarity and protein stability.

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.

Loss of PTEN selectively desensitizes upstream IGF1 and insulin signaling.

Many tumors have chronically elevated activity of PI 3-kinase-dependent signaling pathways, caused largely by oncogenic mutation of PI 3-kinase itself or loss of the opposing tumor suppressor lipid phosphatase, PTEN. Several PI 3-kinase-dependent feedback mechanisms have been identified that may affect the sensitivity of upstream receptor signaling, but the events required to initiate an inhibited state have not been addressed. We show that in a variety of cell types, loss of PTEN via experimental knockdown or in tumor cell lines correlates with a block in insulin-like growth factor 1 (IGF1)/insulin signaling, without affecting the sensitivity of platelet-derived growth factor or epidermal growth factor signaling. These effects on IGF/insulin signaling include a reduction of up to five- to tenfold in IGF-stimulated PI 3-kinase activation, a failure to activate the ERK kinases and, in some cells, reduced expression of insulin receptor substrate 1, and both IGF1 and insulin receptors. These data indicate that chronically elevated PI 3-kinase-dependent signaling to the degree seen in many tumors causes a selective loss of sensitivity in IGF1/insulin signaling that could significantly reduce the selective advantage of deregulated activation of IGF1/IGF1-R signaling in tumor development.

Metabolic switching of PI3K-dependent lipid signals.

The lipid phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10), is the product of a major tumour suppressor gene that antagonizes PI3K (phosphoinositide 3-kinase) signalling by dephosphorylating the 3-position of the inositol ring of PtdIns(3,4,5)P(3). PtdIns(3,4,5)P(3) is also metabolized by removal of the 5-phosphate catalysed by a distinct family of enzymes exemplified by SHIP1 [SH2 (Src homology 2)-containing inositol phosphatase 1] and SHIP2. Mouse knockout studies, however, suggest that PTEN and SHIP2 have profoundly different biological functions. One important reason for this is likely to be that SHIP2 exists in a relatively inactive state until cells are exposed to growth factors or other stimuli. Hence, regulation of SHIP2 is geared towards stimulus dependent antagonism of PI3K signalling. PTEN, on the other hand, appears to be active in unstimulated cells and functions to maintain basal PtdIns(3,4,5)P(3) levels below the critical signalling threshold. We suggest that concomitant inhibition of cysteine-dependent phosphatases, such as PTEN, with activation of SHIP2 functions as a metabolic switch to regulate independently the relative levels of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2).

The regulation of membrane to cytosol partitioning of signalling proteins by phosphoinositides and their soluble headgroups.

Inositol phospholipids [PIs (phosphoinositides)] represent a group of membrane-tethered signalling molecules which differ with respect to the number and distribution of monoester phosphate groups around the inositol ring. They function by binding to proteins which possess one of several domains that bind a particular PI species, often with high affinity and specificity. PH (pleckstrin homology) domains for example possess ligand-binding pockets that are often lined with positively charged residues and which bind PIs with varying degrees of specificity. Several PH domains bind not only PIs, but also their cognate headgroups, many of which occur naturally in cells as relatively abundant cytosolic inositol phosphates. The subcellular distributions of proteins possessing such PH domains are therefore determined by the relative levels of competing membrane-bound and soluble ligands. A classic example of the latter is the PH domain of phospholipase Cdelta1, which binds both phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate. We have shown that the N-terminal PH domain of the Rho family guanine nucleotide-exchange factor, Tiam 1, binds PI ligands promiscuously allowing multiple modes of regulation. We also recently analysed the ligand-binding specificity of the PH domain of PI-dependent kinase 1 and found that it could bind abundant inositol polyphosphates such as inositol hexakisphosphate. This could explain the dual distribution of this key signalling component, which needs to access substrates at both the plasma membrane and in the cytosol.

The regulation of cell migration by PTEN.

In vertebrates, the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) regulates many cellular processes through its PtdIns(3,4,5)P3 lipid phosphatase activity, antagonizing PI3K (phosphoinositide 3-kinase) signalling. Given the important role of PI3Ks in the regulation of directed cell migration and the role of PTEN as an inhibitor of migration, it is somewhat surprising that data now indicate that PTEN is able to regulate cell migration independent of its lipid phosphatase activity. Here, we discuss the role of PTEN in the regulation of cell migration.

Redox regulation of phosphatase function.

Although reactive oxygen species play important roles in cellular physiology as signalling molecules, their molecular targets are largely unknown. A probable group of targets for mediating many of the effects of reactive oxygen species on cell signalling is the large diverse family of cysteine-dependent phosphatases, which includes the protein tyrosine phosphatases. Our work and that of others suggest that the oxidative inactivation of protein and lipid phosphatases plays an important part in signalling, downstream of many cellular stimuli. Future studies should give us a clearer picture of the role of phosphatase inactivation in cellular behaviour and explain how specificity is achieved in redox signalling.

Acute regulation of the tumour suppressor phosphatase, PTEN, by anionic lipids and reactive oxygen species.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a member of the protein tyrosine phosphatase family that is structurally adapted to facilitate the metabolism of 3-phosphoinositide lipid second messengers, especially PtdIns(3,4,5) P (3). Cellular PTEN activity is restrained by the retention of C-terminally phosphorylated enzyme in the cytosol. Dephosphorylation by as yet undefined phosphatases initiates an electrostatic switch which targets PTEN specifically to the plasma membrane, where it binds through multiple positively charged residues in both the C2 and N-terminal domains and is susceptible to feedback regulation through proteolytic degradation. PTEN also forms signalling complexes with PDZ domain-containing adaptors, such as the MAGUK (membrane-associated guanylate kinase) proteins, interactions which appear to be necessary for metabolism of localized pools of PtdIns(3,4,5) P (3) involved in regulating actin cytoskeleton dynamics. TPIP [TPTE (transmembrane phosphatase with tensin homology) and PTEN homologous inositol lipid phosphatase] is a novel gene product which exists in multiply spliced forms. TPIPalpha has PtdIns(3,4,5) P (3) 3-phosphatase activity and is localized to the endoplasmic reticulum, via two transmembrane spanning regions, where it may metabolize PtdIns(3,4,5) P (3) that appears to be unaffected by expressed PTEN. PTEN can be acutely regulated by oxidative stress and by endogenously produced reactive oxygen species. This mechanism provides a novel means to stimulate phosphoinositide 3-kinase-dependent signalling pathways, which may be important in circumstances where PtdIns(3,4,5) P (3) and oxidants are produced concurrently.

TPIP: a novel phosphoinositide 3-phosphatase.

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor is a phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] 3-phosphatase that plays a critical role in regulating many cellular processes by antagonizing the phosphoinositide 3-kinase signalling pathway. We have identified and characterized two human homologues of PTEN, which differ with respect to their subcellular localization and lipid phosphatase activities. The previously cloned, but uncharacterized, TPTE (transmembrane phosphatase with tensin homology) is localized to the plasma membrane, but lacks detectable phosphoinositide 3-phosphatase activity. TPIP (TPTE and PTEN homologous inositol lipid phosphatase) is a novel phosphatase that occurs in several differentially spliced forms of which two, TPIP alpha and TPIP beta, appear to be functionally distinct. TPIP alpha displays similar phosphoinositide 3-phosphatase activity compared with PTEN against PtdIns(3,4,5)P(3), PtdIns(3,5)P(2), PtdIns(3,4)P(2) and PtdIns(3)P, has N-terminal transmembrane domains and appears to be localized on the endoplasmic reticulum. This is unusual as most signalling-lipid-metabolizing enzymes are not integral membrane proteins. TPIP beta, however, lacks detectable phosphatase activity and is cytosolic. TPIP has a wider tissue distribution than the testis-specific TPTE, with specific splice variants being expressed in testis, brain and stomach. TPTE and TPIP do not appear to be functional orthologues of the Golgi-localized and more distantly related murine PTEN2. We suggest that TPIP alpha plays a role in regulating phosphoinositide signalling on the endoplasmic reticulum, and might also represent a tumour suppressor and functional homologue of PTEN in some tissues.

Antagonism of PI 3-kinase-dependent signalling pathways by the tumour suppressor protein, PTEN.

The tumour suppressor protein, PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a member of the mixed function, serine/threonine/tyrosine phosphatase subfamily of protein phosphatases. Its physiological substrates, however, are primarily 3-phosphorylated inositol phospholipids, which are products of phosphoinositide 3-kinases. PTEN thus antagonizes PI 3-kinase-dependent signalling pathways, which explains to a large extent its tumour suppressor status. We have examined the kinetic behaviour, substrate specificity and regulation of PTEN both in vitro and in a variety of cellular models. Although PTEN can utilize both phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] and its water-soluble headgroup, inositol 1,3,4,5-tetrakisphosphate, as substrates, it displays classical features of interfacial catalysis, which greatly favour the lipid substrate (by as much as 1000-fold as judged by K(cat)/K(m) values). Expression of PTEN in U87 cells (which lack endogenous PTEN) and measuring the levels of all known 3-phosphorylated lipids suggests that phosphatidylinositol 3,4-bisphosphate and PtdIns(3,4,5)P(3) are both substrates, but that phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate are not. PTEN binds to several PDZ-domain-containing proteins via a consensus sequence at its extreme C-terminus. Disruption of targeting to PDZ-domain proteins selectively blocks some PTEN functions, but not others, suggesting the existence of spatially localized, functionally dedicated pools of signalling lipids. We have also shown recently that PTEN expression is controlled at the transcriptional level and is profoundly upregulated by peroxisome proliferator activated receptor gamma agonists, thereby providing possible implications for these drugs in diabetes, inflammation and cancer.

Regulation of clathrin-coated vesicle formation.

The formation of clathrin-coated pits at the plasma membrane requires the concerted action of many different molecules. The real challenge lies in determining the hierarchy of these interactions. We are using assays in both intact and permeabilized cells to dissect the temporal requirements for clathrin-coated vesicle formation, and also to examine the role of phosphorylation of the coat proteins.

Targeting mutants of PTEN reveal distinct subsets of tumour suppressor functions.

The tumour suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase which can antagonize the phosphoinositide 3-kinase (PI 3-kinase) signalling pathway, promoting apoptosis and inhibiting cell-cycle progression and cell motility. We show that very little cellular PTEN is associated with the plasma membrane, but that artificial membrane-targeting of PTEN enhances its inhibition of signalling to protein kinase B (PKB). Evidence for potential targeting of PTEN to the membrane through PDZ domain-mediated protein-protein interactions led us to use a PTEN enzyme with a deletion of the C-terminal PDZ-binding sequence, that retains full phosphatase activity against soluble substrates, and to analyse the efficiency of this mutant in different cellular assays. The extreme C-terminal PDZ-binding sequence was dispensable for the efficient down-regulation of cellular PtdIns(3,4,5)P3 levels and a number of PI 3-kinase-dependent signalling activities, including PKB and p70S6K. However, the PDZ-binding sequence was required for the efficient inhibition of cell spreading. The data show that a PTEN mutation, similar to those found in some tumours, affects some functions of the protein but not others, and implicate the deregulation of PTEN-dependent processes other than PKB activation in the development of some tumours. Significantly, this hypothesis is supported by data showing low levels of PKB phosphorylation in a glioblastoma sample carrying a mutation in the extreme C-terminus of PTEN compared with tumours carrying phosphatase-inactivating mutations of the enzyme. Our data show that deregulation of PKB is not a universal feature of tumours carrying PTEN mutations and implicate other processes that may be deregulated in these tumours.

Tumor suppressor and anti-inflammatory actions of PPARgamma agonists are mediated via upregulation of PTEN.

The PTEN tumor suppressor gene modulates several cellular functions, including cell migration, survival, and proliferation [1] by antagonizing phosphatidylinositol 3-kinase (PI 3-kinase)-mediated signaling cascades. Mechanisms by which the expression of PTEN is regulated are, however, unclear. The ligand-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) [2] has been shown to regulate differentiation and/or cell growth in a number of cell types [3, 4, 5], which has led to the suggestion that PPARgamma, like PTEN [1, 6], could act as a tumor suppressor. PPARgamma has also been implicated in anti-inflammatory responses [7, 8], although downstream mediators of these effects are not well defined. Here, we show that the activation of PPARgamma by its selective ligand, rosiglitazone, upregulates PTEN expression in human macrophages, Caco2 colorectal cancer cells, and MCF7 breast cancer cells. This upregulation correlated with decreased PI 3-kinase activity as measured by reduced phosphorylation of protein kinase B. One consequence of this was that rosiglitazone treatment reduced the proliferation rate of Caco2 and MCF7 cells. Antisense-mediated disruption of PPARgamma expression prevented the upregulation of PTEN that normally accompanies monocyte differentiation and reduced the proportion of macrophages undergoing apoptosis, while electrophoretic mobility shift assays showed that PPARgamma is able to bind two response elements in the genomic sequence upstream of PTEN. Our results demonstrate a role for PPARgamma in regulating PI 3-kinase signaling by modulating PTEN expression in inflammatory and tumor-derived cells.

The role of dynamin and its binding partners in coated pit invagination and scission.

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

Ceramide impairs the insulin-dependent membrane recruitment of protein kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells.

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, C2-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 C2-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 micromol/l) for 2 h led to a complete loss of insulin-stimulated 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-phosphoinositide-dependent kinase-1, or to impaired binding of phosphatidylinositol 3,4,5 triphosphate (PtdIns(3,4,5)P3) 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)P3 is necessary but not sufficient for protein kinase B activation in this system.

Beta1-integrin and PTEN control the phosphorylation of protein kinase C.

Phosphorylation of protein kinase C (PKC) provides an amplitude control that operates in conjunction with allosteric effectors. Under many conditions, PKC isotypes appear to be highly phosphorylated; however, the cellular inputs that maintain these phosphorylations are not characterized. In the present work, it is shown that there is a differential phosphorylation of PKCdelta in adherent versus suspension cultures of transfected HEK-293 cells. It is established that integrin activation is sufficient to trigger PKCdelta phosphorylation and that this signals through phosphoinositide 3-kinase (PI3-kinase) to stimulate the phosphorylation of two sites, T505 and S662. The loss of signal input to PKCdelta in suspension culture is dependent on the tumour suppressor gene PTEN, which encodes a bi-functional phosphotyrosine/phosphoinositide 3-phosphate phosphatase. In the PTEN(-/-) UM-UC-3 bladder carcinoma cell line grown in suspension, transfected PKCdelta no longer accumulates in a dephospho-form on serum removal. By contrast, in a UM-UC-3-derivative cell line stably expressing PTEN, PKCdelta does become dephosphorylated under these conditions. Employing the PTEN Gly(129)-->Glu mutant, which is selectively defective in lipid phosphatase activity, it was established that it is the lipid phosphatase activity that controls PKCdelta phosphorylation. The evidence indicates that PKCdelta phosphorylation and its latent activity are maintained in serum-deprived adherent cultures through integrin-matrix interactions. This control acts through a pathway involving a lipid product of PI3-kinase in a manner that can be suppressed by PTEN.

Activation of phospholipase D by metabotropic glutamate receptor agonists in rat cerebrocortical synaptosomes.

The pharmacological profile of metabotropic glutamate receptor (mGluR) activation of phospholipase D (PLD), and the associated signalling pathways, were examined in rat cerebrocortical synaptosomes. The assay was conducted using a transphosphatidylation reaction in synaptosomes which were pre-labelled with either [(3)H]-arachidonic acid or [(32)P]-orthophosphate. The mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S, 3R-ACPD) and (RS)-3,5-dihydroxyphenylglycine (DHPG), both activated PLD, while phorbol 12,13-dibutyrate (PDBu) treatment caused receptor-independent activation of PLD and had an additive effect on 1S,3R-ACPD induced PLD activity. A protein kinase C (PKC) inhibitor, GF109203X, failed to antagonize mGluR receptor-coupled PLD activity. We could not detect any increase in the products of PI (phosphoinositide)-specific phospholipase C (PI-PLC), inositol(1,4, 5)trisphosphate or diacylglycerol, by 1S, 3R-ACPD at 15 s. However, diacylglycerol increased monophasically in response to mGluR agonists and remained elevated for at least 15 min. Phosphatidic acid phosphohydrolase (PAP) activity, which converts PA to DAG, was present in the synaptosomes. These data suggest that, in rat cerebrocortical synaptosomes, the 1S,3R-ACPD-sensitive mGluR is coupled to PLD through a mechanism that is independent of both PKC and PI-PLC.

Regulation of the Rac1-specific exchange factor Tiam1 involves both phosphoinositide 3-kinase-dependent and -independent components.

The small GTPase Rac1 is involved in regulating membrane ruffling, gene transcription, cell-cycle progression and cell transformation, and some of these events are blocked by inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Moreover, Rac1 can be activated by several guanine nucleotide exchange factors, which facilitate the release of GDP. We therefore investigated the ability of PI 3-kinase lipid products to regulate Tiam1, a Rac1-specific exchange factor. Tiam1 bound to polyphosphorylated inositol lipids in the rank order PtdIns(3,4,5)P(3)>PtdIns(3,4)P(2) >>PtdIns(4,5)P(2), and this binding could be attributed to the N-terminal pleckstrin-homology (N-PH) domain. Both PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) enhanced Tiam1 guanine nucleotide exchange activity in vitro, but PtdIns(4,5)P(2) had no effect. Co-expression of a constitutively active PI 3-kinase with Tiam1 increased the amount of GTP-bound Rac1 in vivo, a response which required the N-PH domain of Tiam1. Ectopic expression of Tiam1 caused membrane ruffling in Swiss 3T3 cells that was characterized by wortmannin-sensitive and -insensitive components, which required the N-PH domain and the C-terminal PH domain of Tiam1 respectively. These results reveal novel facets of Tiam1-dependent regulation of Rac1 function.

Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities.

The second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is generated by the action of phosphoinositide 3-kinase (PI 3-kinase), and regulates a plethora of cellular processes. An approach for dissecting the mechanisms by which these processes are regulated is to identify proteins that interact specifically with PtdIns(3,4,5)P(3). The pleckstrin homology (PH) domain has become recognized as the specialized module used by many proteins to interact with PtdIns(3,4,5)P(3). Recent work has led to the identification of a putative phosphatidylinositol 3,4,5-trisphosphate-binding motif (PPBM) at the N-terminal regions of PH domains that interact with this lipid. We have searched expressed sequence tag databases for novel proteins containing PH domains possessing a PPBM. Surprisingly, many of the PH domains that we identified do not bind PtdIns(3,4,5)P(3), but instead possess unexpected and novel phosphoinositide-binding specificities in vitro. These include proteins possessing PH domains that interact specifically with PtdIns(3,4)P(2) [TAPP1 (tandem PH-domain-containing protein-1) and TAPP2], PtdIns4P [FAPP1 (phosphatidylinositol-four-phosphate adaptor protein-1)], PtdIns3P [PEPP1 (phosphatidylinositol-three-phosphate-binding PH-domain protein-1) and AtPH1] and PtdIns(3,5)P(2) (centaurin-beta2). We have also identified two related homologues of PEPP1, termed PEPP2 and PEPP3, that may also interact with PtdIns3P. This study lays the foundation for future work to establish the phospholipid-binding specificities of these proteins in vivo, and their physiological role(s).

Role of phosphatidylinositol 3-kinase and specific protein kinase B isoforms in the suppression of apoptosis mediated by the Abelson protein-tyrosine kinase.

Leukemogenic oncogenes, such as the Abelson protein-tyrosine kinases (PTK), disrupt the normal regulation of survival, proliferation, and differentiation in hemopoietic progenitor cells. In the absence of cytokines, hemopoietic progenitor cells die by apoptosis. Abl PTKs mediate suppression of this apoptotic response leading to aberrant survival. To investigate the mechanism of Abl PTK action, we have used an interleukin-3-dependent murine mast cell line that expresses a temperature-sensitive form of the v-ABL PTK, which is active at the permissive temperature of 32 degrees C and inactive at 39 degrees C. At the permissive temperature, these cells are resistant to apoptosis induced both by the withdrawal of the hemopoietic growth factor (interleukin-3) and the addition of cytotoxic drugs. We demonstrate that v-Abl associates with and stimulates activation of phosphatidylinositol 3-kinase (PI3K) and, crucially, that this activation results in enhanced cellular levels of the mass of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Activation of PI3K leads to enhanced activity of PKB and increased levels of the anti-apoptotic protein Bcl-X(L). Transfection of cells with a dominant negative PKB reduces both the Abl-stimulated PKB activity and the survival effect conferred by activation of this oncogene. Thus, PI3K and PKB are required for the anti-apoptotic effects of Abl PTK.