Phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) is a key molecule involved in cell growth signaling. We demonstrated that overexpression of PTEN, a putative tumor suppressor, reduced insulin-induced <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> production in human 293 cells without effecting insulin-induced phosphoinositide 3-kinase activation. Further, transfection of the catalytically inactive mutant of PTEN (C124S) caused <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> accumulation in the absence of insulin stimulation. Purified recombinant PTEN catalyzed dephosphorylation of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, specifically at position 3 on the inositol ring. PTEN also exhibited 3-phosphatase activity toward inositol 1,<em>3,4,5</em>-tetrakisphosphate. Our results raise the possibility that PTEN acts in vivo as a phosphoinositide 3-phosphatase by regulating <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> levels. As expected, the C124S mutant of PTEN was incapable of catalyzing dephosphorylation of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> consistent with the mechanism observed in protein-tyrosine phosphatase-catalyzed reactions.

BACKGROUND

Protein kinase B (PKB), also known as c-Akt, is activated rapidly when mammalian cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies 'downstream' of phosphoinositide 3-kinase on intracellular signalling pathways. We recently showed that insulin or insulin-like growth factor 1 induce the phosphorylation of PKB at two residues, Thr308 and Ser473. The phosphorylation of both residues is required for maximal activation of PKB. The kinases that phosphorylate PKB are, however, unknown.

RESULTS

We have purified 500 000-fold from rabbit skeletal muscle extracts a protein kinase which phosphorylates PKBalpha at Thr308 and increases its activity over 30-fold. We tested the kinase in the presence of several inositol phospholipids and found that only low micromolar concentrations of the D enantiomers of either phosphatidylinositol <em>3,4,5</em>-triphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) or <em>PtdIns</em>(3,4)P2 were effective in potently activating the kinase, which has been named <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-dependent protein kinase-1 (PDK1). None of the inositol phospholipids tested activated or inhibited PKBalpha or induced its phosphorylation under the conditions used. PDK1 activity was not affected by wortmannin, indicating that it is not likely to be a member of the phosphoinositide 3-kinase family. CONLCUSIONS: PDK1 is likely to be one of the protein kinases that mediate the activation of PKB by insulin and growth factors. PDK1 may, therefore, play a key role in mediating many of the actions of the second messenger(s) <em>PtdIns</em>(3,4, 5)<em>P3</em> and/or <em>PtdIns</em>(3,4)P2.

Eleven distinct isoforms of phosphoinositide-specific phospholipase C (PLC), which are grouped into four subfamilies (beta, gamma, delta, and epsilon), have been identified in mammals. These isozymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate [<em>PtdIns</em>(4,5)P2] to inositol 1,4,5-trisphosphate and diacylglycerol in response to the activation of more than 100 different cell surface receptors. All PLC isoforms contain X and Y domains, which form the catalytic core, as well as various combinations of regulatory domains that are common to many other signaling proteins. These regulatory domains serve to target PLC isozymes to the vicinity of their substrate or activators through protein-protein or protein-lipid interactions. These domains (with their binding partners in parentheses or brackets) include the pleckstrin homology (PH) domain [<em>PtdIns</em>(3)P, beta gamma subunits of G proteins] and the COOH-terminal region including the C2 domain (GTP-bound alpha subunit of Gq) of PLC-beta; the PH domain [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>] and Src homology 2 domain [tyrosine-phosphorylated proteins, <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>] of PLC-gamma; the PH domain [<em>PtdIns</em>(4,5)P2] and C2 domain (Ca2+) of PLC-delta; and the Ras binding domain (GTP-bound Ras) of PLC-epsilon. The presence of distinct regulatory domains in PLC isoforms renders them susceptible to different modes of activation. Given that the partners that interact with these regulatory domains of PLC isozymes are generated or eliminated in specific regions of the cell in response to changes in receptor status, the activation and deactivation of each PLC isoform are likely highly regulated processes.

Protein kinase B (PKB) is a proto-oncogene that is activated in signaling pathways initiated by phosphoinositide 3-kinase. Chromatographic separation of brain cytosol revealed a kinase activity that phosphorylated and activated PKB only in the presence of phosphatidylinositol-<em>3,4,5</em>-trisphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>]. Phosphorylation occurred exclusively on threonine-308, a residue implicated in activation of PKB in vivo. <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> was determined to have a dual role: Its binding to the pleckstrin homology domain of PKB was required to allow phosphorylation by the upstream kinase and it directly activated the upstream kinase.

Protein kinase B (PKB) is activated in response to phosphoinositide 3-kinases and their lipid products phosphatidylinositol 3,4, 5-trisphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>] and <em>PtdIns</em>(3,4)P2 in the signaling pathways used by a wide variety of growth factors, antigens, and inflammatory stimuli. PKB is a direct target of these lipids, but this regulation is complex. The lipids can bind to the pleckstrin homologous domain of PKB, causing its translocation to the membrane, and also enable upstream, Thr308-directed kinases to phosphorylate and activate PKB. Four isoforms of these PKB kinases were purified from sheep brain. They bound <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and associated with lipid vesicles containing it. These kinases contain an NH2-terminal catalytic domain and a COOH-terminal pleckstrin homologous domain, and their heterologous expression augments receptor activation of PKB, which suggests they are the primary signal transducers that enable <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> or <em>PtdIns</em>- (3,4)P2 to activate PKB and hence to control signaling pathways regulating cell survival, glucose uptake, and glycogen metabolism.

The interaction of insulin and growth factors with their receptors on the outside surface of a cell, leads to the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and generation of the phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) second messenger at the inner surface of the cell membrane. One of the most studied signalling events controlled by <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, comprises the activation of a group of AGC family protein kinases, including isoforms of protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-induced protein kinase (SGK) and protein kinase C (PKC), which play crucial roles in regulating physiological processes relevant to metabolism, growth, proliferation and survival. Here, we review recent biochemical, genetic and structural studies on the 3-phosphoinositide-dependent protein kinase-1 (PDK1), which phosphorylates and activates the AGC kinase members regulated by PI 3-kinase. We also discuss whether inhibitors of PDK1 might have chemotherapeutic potential in the treatment of cancers in which the PDK1-regulated AGC kinases are constitutively activated.

The roles of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) in chemoattractant-elicited responses were studied in mice lacking these key enzymes. PI3Kgamma was required for chemoattractant-induced production of phosphatidylinositol <em>3,4,5</em>-trisphosphate [<em>PtdIns</em> (<em>3,4,5</em>)<em>P3</em>] and has an important role in chemoattractant-induced superoxide production and chemotaxis in mouse neutrophils and in production of T cell-independent antigen-specific antibodies composed of the immunoglobulin lambda light chain (TI-IglambdaL). The study of the mice lacking PLC-beta2 and -beta3 revealed that the PLC pathways have an important role in chemoattractant-mediated production of superoxide and regulation of protein kinases, but not chemotaxis. The PLC pathways also appear to inhibit the chemotactic activity induced by certain chemoattractants and to suppress TI-IglambdaL production.

Domains or modules known to bind phosphoinositides have increased dramatically in number over the past few years, and are found in proteins involved in intracellular trafficking, cellular signaling, and cytoskeletal remodeling. Analysis of lipid binding by these domains and its structural basis has provided significant insight into the mechanism of membrane recruitment by the different cellular phosphoinositides. Domains that target only the rare (3-phosphorylated) phosphoinositides must bind with very high affinity, and with exquisite specificity. This is achieved solely by headgroup interactions in the case of certain pleckstrin homology (PH) domains [which bind <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and/or <em>PtdIns</em>(3,4)P2], but requires an additional membrane-insertion and/or oligomerization component in the case of the <em>PtdIns</em>(3)P-targeting phox homology (PX) and FYVE domains. Domains that target <em>PtdIns</em>(4,5)P2, which is more abundant by some 25-fold, do not require the same stringent affinity and specificity characteristics, and tend to be more diverse in structure. The mode of phosphoinositide binding by different domains also appears to reflect their distinct functions. For example, pleckstrin homology domains that serve as simple targeting domains recognize only phosphoinositide headgroups. By contrast, certain other domains, notably the epsin ENTH domain, appear to promote bilayer curvature by inserting into the membrane upon binding.

It is now accepted that activation of Class I PI3Ks (phosphoinositide 3-kinases) is one of the most important signal transduction pathways used by cell-surface receptors to control intracellular events. The receptors which access this pathway include those that recognize growth factors, hormones, antigens and inflammatory stimuli, and the cellular events known to be regulated include cell growth, survival, proliferation and movement. We have learnt a great deal about the family of Class I PI3K enzymes themselves and the structural adaptations which allow a variety of cell-surface receptors to regulate their activity. Class I PI3Ks synthesize the phospholipid <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> in the membranes in which they are activated, and it is now accepted that <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and its dephosphorylation product <em>PtdIns</em>(3,4)P2 are messenger molecules which regulate the localization and function of multiple effectors by binding to their specific PH (pleckstrin homology) domains. The number of direct <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>/<em>PtdIns</em>(3,4)P2 effectors which exist, even within a single cell, creates an extremely complex signalling web downstream of PI3K activation. Some key players are beginning to emerge, however, linking PI3K activity to specific cellular responses. These include small GTPases for the Rho and Arf families which regulate the cytoskeletal and membrane rearrangements required for cell movement, and PKB (protein kinase B), which has important regulatory inputs into the regulation of cell-cycle progression and survival. The importance of the PI3K signalling pathway in regulating the balance of decisions in cell growth, proliferation and survival is clear from the prevalence of oncogenes (e.g. PI3Kalpha) and tumour suppressors [e.g. the <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> 3-phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10)] found in this pathway. The recent availability of transgenic mouse models with engineered defects in Class I PI3K signalling pathways, and the development of PI3K isoform-selective inhibitors by both academic and pharmaceutical research has highlighted the importance of specific isoforms of PI3K in whole-animal physiology and pathology, e.g. PI3Kalpha in growth and metabolic regulation, PI3Kbeta in thrombosis, and PI3Kdelta and PI3Kgamma in inflammation and asthma. Thus the Class I PI3K signalling pathway is emerging as an exciting new area for the development of novel therapeutics.

Rac, a member of the Rho family of monomeric GTPases, is an integrator of intracellular signaling in a wide range of cellular processes. We have purified a <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-sensitive activator of Rac from neutrophil cytosol. It is an abundant, 185 kDa guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially, and synergistically activated by <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and Gbetagammas both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a coincidence detector in <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and Gbetagamma signaling pathways that is particularly adapted to function downstream of heterotrimeric G proteins in neutrophils.

The <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-dependent activation of protein kinase B (PKB) by 3-phosphoinositide-dependent protein kinases-1 and -2 (PDK1 and PDK2 respectively) is a key event in mediating the effects of signals that activate <em>PtdIns</em> 3-kinase. The catalytic domain of serum- and glucocorticoid-regulated protein kinase (SGK) is 54% identical with that of PKB and, although lacking the <em>PtdIns</em>(3,4, 5)<em>P3</em>-binding pleckstrin-homology domain, SGK retains the residues that are phosphorylated by PDK1 and PDK2, which are Thr256 and Ser422 in SGK. Here we show that PDK1 activates SGK in vitro by phosphorylating Thr256. We also show that, in response to insulin-like growth factor-1 (IGF-1) or hydrogen peroxide, transfected SGK is activated in 293 cells via a <em>PtdIns</em> 3-kinase-dependent pathway that involves the phosphorylation of Thr256 and Ser422. The activation of SGK by PDK1 in vitro is unaffected by <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, abolished by the mutation of Ser422 to Ala, and greatly potentiated by mutation of Ser422 to Asp (although this mutation does not activate SGK itself). Consistent with these findings, the Ser422Asp mutant of SGK is activated by phosphorylation (probably at Thr256) in unstimulated 293 cells, and activation is unaffected by inhibitors of <em>PtdIns</em> 3-kinase. Our results are consistent with a model in which activation of SGK by IGF-1 or hydrogen peroxide is initiated by a <em>PtdIns</em>(3,4, 5)<em>P3</em>-dependent activation of PDK2, which phosphorylates Ser422. This is followed by the <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-independent phosphorylation at Thr256 that activates SGK, and is catalysed by PDK1. Like PKB, SGK preferentially phosphorylates serine and threonine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs, and SGK and PKB inactivate glycogen synthase kinase-3 similarly in vitro and in co-transfection experiments. These findings raise the possibility that some physiological roles ascribed to PKB on the basis of the overexpression of constitutively active PKB mutants might be mediated by SGK.

Recent advances in our understanding of the role of inositides in cell signalling have led to the central hypothesis that a receptor-stimulated phosphodiesteratic hydrolysis of phosphatidylinositol 4,5-bisphosphate (<em>PtdIns</em>(4,5)P2) results in the formation of two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)<em>P3</em>). The existence of another pathway of inositide metabolism was first suggested by the discovery that a novel inositol trisphosphate, Ins(1,3,4)<em>P3</em>, is formed in stimulated tissues; the metabolic kinetics of Ins(1,3,4)<em>P3</em> are entirely different from those of Ins(1,4,5)<em>P3</em> (refs 6, 7). The probable route of formation of Ins(1,3,4)<em>P3</em> was recently shown to be via a 5-dephosphorylation of inositol 1,<em>3,4,5</em>-tetrakisphosphate (Ins(1,<em>3,4,5</em>)P4), a compound which is rapidly formed on muscarinic stimulation of brain slices, and which can be readily converted to Ins(1,3,4)<em>P3</em> by a 5-phosphatase in red blood cell membranes. However, the source of Ins(1,<em>3,4,5</em>)P4 is unclear, and an attempt to detect a possible parent lipid, phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>), was unsuccessful. The recent discovery that the higher phosphorylated forms of inositol (InsP5 and InsP6) also exist in animal cells suggested that inositol phosphate kinases might not be confined to plant and avian tissues, and here we show that a variety of animal tissues contain an active and specific Ins(1,4,5)<em>P3</em> 3-kinase. We therefore suggest that an inositol tris/tetrakisphosphate pathway exists as an alternative route to the dephosphorylation of Ins(1,4,5)<em>P3</em>. The function of this novel pathway is unknown.

BACKGROUND

Protein kinase C zeta (PKC zeta) is a member of the PKC family of enzymes and is involved in a wide range of physiological processes including mitogenesis, protein synthesis, cell survival and transcriptional regulation. PKC zeta has received considerable attention recently as a target of phosphoinositide 3-kinase (PI 3-kinase), although the mechanism of PKC zeta activation is, as yet, unknown. Recent reports have also shown that the phosphoinositide-dependent protein kinase-1 (PDK-1), which binds with high affinity to the PI 3-kinase lipid product phosphatidylinositol-<em>3,4,5</em>-trisphosphate (<em>Ptdins</em>-<em>3,4,5</em>-<em>P3</em>), phosphorylates and potently activates two other PI 3-kinase targets, the protein kinases Akt/PKB and p70S6K. We therefore investigated whether PDK-1 is the kinase that activates PKC zeta.

RESULTS

In vivo, PI 3-kinase is both necessary and sufficient to activate PKC zeta. PDK-1 phosphorylates and activates PKC zeta in vivo, and we have shown that this is due to phosphorylation of threonine 410 in the PKC zeta activation loop. In vitro, PDK-1 phosphorylates and activates PKC zeta in a <em>Ptdins</em>-<em>3,4,5</em>-<em>P3</em>-enhanced manner. PKC zeta and PDK-1 are associated in vivo, and membrane targeting of PKC zeta renders it constitutively active in cells.

CONCLUSIONS

Our results have identified PDK-1 as the kinase that phosphorylates and activates PKC zeta in the PI 3-kinase signaling pathway. This phosphorylation and activation of PKC zeta by PDK-1 is enhanced in the presence of Ptdins-3,4-5-P3. Consistent with the notion that PKCs are enzymes that are regulated at the plasma membrane, a membrane-targeted PKC zeta is constitutively active in the absence of agonist stimulation. The association between PKC zeta and PDK-1 reveals extensive cross-talk between enzymes in the PI 3-kinase signaling pathway.

PTEN, a tumor suppressor located at chromosome 10q23, is mutated in a variety of sporadic cancers and in two autosomal dominant hamartoma syndromes. PTEN is a phosphatase which dephosphorylates phosphatidylinositol (<em>3,4,5</em>)-triphosphate (<em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>), an important intracellular second messenger, lowering its level within the cell. By dephosphorylating <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>, PTEN acts in opposition to phosphatidylinositol 3-kinase (PI3K), which has a pivotal role in the creation of <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>. <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> is necessary for the activation of Akt, a serine/threonine kinase involved in cell growth and survival. By blocking the activation of Akt, PTEN regulates cellular processes such as cell cycling, translation, and apoptosis. In this review, we will discuss the identification of PTEN, its mutational status in cancer, its role as a regulator of PI3K, and its domain structure.

Phosphoinositide 3-kinase (PI3K), PTEN and localized phosphatidylinositol (<em>3,4,5</em>)-trisphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>] play key roles in chemotaxis, regulating cell motility by controlling the actin cytoskeleton in Dictyostelium and mammalian cells. <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, produced by PI3K, acts via diverse downstream signaling components, including the GTPase Rac, Arf-GTPases and the kinase Akt (PKB). It has become increasingly apparent, however, that chemotaxis results from an interplay between the PI3K-PTEN pathway and other parallel pathways in Dictyostelium and mammalian cells. In Dictyostelium, the phospholipase PLA2 acts in concert with PI3K to regulate chemotaxis, whereas phospholipase C (PLC) plays a supporting role in modulating PI3K activity. In adenocarcinoma cells, PLC and the actin regulator cofilin seem to provide the direction-sensing machinery, whereas PI3K might regulate motility.

Phosphotidylinositol (<em>PtdIns</em>) signaling is tightly regulated both spatially and temporally by subcellularly localized <em>PtdIns</em> kinases and phosphatases that dynamically alter downstream signaling events. Joubert syndrome is characterized by a specific midbrain-hindbrain malformation ('molar tooth sign'), variably associated retinal dystrophy, nephronophthisis, liver fibrosis and polydactyly and is included in the newly emerging group of 'ciliopathies'. In individuals with Joubert disease genetically linked to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and <em>PtdIns</em>(4,5)P2. Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular <em>PtdIns</em> ratios. INPP5E localized to cilia in major organs affected by Joubert syndrome, and mutations promoted premature destabilization of cilia in response to stimulation. These data link <em>PtdIns</em> signaling to the primary cilium, a cellular structure that is becoming increasingly recognized for its role in mediating cell signals and neuronal function.

Inositol phospholipids play multiple roles in cell signalling systems. Two widespread eukaryotic phosphoinositide-based signal transduction mechanisms, phosphoinositidase C-catalysed phosphatidylinositol-4,5-bisphosphate (<em>PtdIns</em>(4,5)P2) hydrolysis and 3-OH kinase-catalysed <em>PtdIns</em>(4,5)P2 phosphorylation, make the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)<em>P3</em>) sn-1,2-diacylglycerol and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. In addition, <em>PtdIns</em>(4,5)P2 and <em>PtdIns</em>3P have been implicated in exocytosis and membrane trafficking. We now show that when the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are hyperosmotically stressed, they rapidly synthesize phosphatidylinositol-3,5-bisphosphate (<em>PtdIns</em>(3,5)P2) by a process that involves activation of a <em>PtdIns</em>3P 5-OH kinase. This <em>PtdIns</em>(3,5)P2 accumulation only occurs in yeasts that have an active vps34-encoded <em>PtdIns</em> 3-OH kinase, showing that this latter kinase makes the <em>PtdIns</em>3P needed for <em>PtdIns</em>(3,5)P2 synthesis and indicating that <em>PtdIns</em>(3,5)P2 may have a role in sorting vesicular proteins. <em>PtdIns</em>(3,5)P2 is also present in mammalian and plant cells: in monkey Cos-7 cells, its labelling is inversely related to the external osmotic pressure. The stimulation of a <em>PtdIns</em>3P 5-OH kinase-catalysed synthesis of <em>PtdIns</em>(3,5)P2, a molecule that might be a new type of phosphoinositide 'second messenger, thus appears to be central to a widespread and previously uncharacterized regulatory pathway.

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that function both as signal transducers downstream of cell-surface receptors and in constitutive intracellular membrane and protein trafficking pathways. All PI3Ks are dual-specificity enzymes with a lipid kinase activity which phosphorylates phosphoinositides at the 3-hydroxyl, and a protein kinase activity. The products of PI3K-catalysed reactions, phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>), <em>PtdIns</em>(3,4)P2 and <em>PtdIns</em>(3)P, are second messengers in a variety of signal transduction pathways, including those essential to cell proliferation, adhesion, survival, cytoskeletal rearrangement and vesicle trafficking. Here we report the 2.2 A X-ray crystallographic structure of the catalytic subunit of PI3Kgamma, the class I enzyme that is activated by heterotrimeric G-protein betagamma subunits and Ras. PI3Kgamma has a modular organization centred around a helical-domain spine, with C2 and catalytic domains positioned to interact with phospholipid membranes, and a Ras-binding domain placed against the catalytic domain where it could drive allosteric activation of the enzyme.

Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cases by binding specific phosphoinositides. We describe use of a convenient "dot-blot" approach to screen 10 different PH domains for those that recognize particular phosphoinositides. Each PH domain bound phosphoinositides in the assay, but only two (from phospholipase C-delta1 and Grp1) showed clear specificity for a single species. Using soluble inositol phosphates, we show that the Grp1 PH domain (originally cloned on the basis of its phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) binding) binds specifically to D-myo-inositol 1,<em>3,4,5</em>-tetrakisphosphate (Ins(1,<em>3,4,5</em>)P4) (the <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> headgroup) with KD = 27.3 nM, but binds D-myo-inositol 1,3,4-trisphosphate (Ins(1,3,4)<em>P3</em>) or D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)<em>P3</em>) over 80-fold more weakly. We show that this specificity allows localization of the Grp1 PH domain to the plasma membrane of mammalian cells only when phosphatidylinositol 3-kinase (PI 3-K) is activated. The presence of three adjacent equatorial phosphate groups was critical for inositol phosphate binding by the Grp1 PH domain. By contrast, another PH domain capable of PI 3-K-dependent membrane recruitment (encoded by EST684797) does not distinguish Ins(1,3,4)<em>P3</em> from Ins(1,<em>3,4,5</em>)<em>P3</em> (binding both with very high affinity), despite selecting strongly against Ins(1,4,5)<em>P3</em>. The remaining PH domains tested appear significantly less specific for particular phosphoinositides. Together with data presented in the literature, our results suggest that many PH domains bind similarly to multiple phosphoinositides (and in some cases phosphatidylserine), and are likely to be regulated in vivo by the most abundant species to which they bind. Thus, using the same simple approach to study several PH domains simultaneously, our studies suggest that highly specific phosphoinositide binding is a characteristic of relatively few cases.

Signaling roles for phosphoinositides that involve their regulated hydrolysis to generate second messengers have been well characterized. Recent work has revealed additional signaling roles for phosphoinositides that do not involve their hydrolysis. <em>PtdIns</em> 3-P, <em>PtdIns</em> <em>3,4,5</em>-<em>P3</em>, and <em>PtdIns</em> 4,5-P2 function as site-specific signals on membranes that recruit and/or activate proteins for the assembly of spatially localized functional complexes. A large number of phosphoinositide-binding proteins have been identified as the potential effectors for phosphoinositide signals. Common themes of localized signal generation and the spatially localized recruitment of effector proteins appear to underlie mechanisms employed in signal transduction, cytoskeletal, and membrane trafficking events.

Phosphoinositide kinases (PI3Ks) play an important role in mitogenic signaling and cell survival, cytoskeletal remodeling, metabolic control and vesicular trafficking. Here we summarize the structure-function relationships delineating the activation process of class I PI3Ks involving various domains of adapter subunits, Ras, and interacting proteins. The resulting product, <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, targets Akt/protein kinase B (PKB), Bruton's tyrosine kinase (Btk), phosphoinositide-dependent kinases (PDK), integrin-linked kinase (ILK), atypical protein kinases C (PKC), phospholipase Cgamma and more. Surface receptor-activated PI3Ks function in mammals, insects, nematodes and slime mold, but not yeast. While many members of the class II family have been identified and characterized biochemically, it is presently unknown how these C2-domain containing PI3Ks are activated, and which PI substrate they phosphorylate in vivo. <em>PtdIns</em> 3-P is produced by Vps34p/class III PI3Ks and operates via the <em>PtdIns</em> 3-P-binding proteins early endosomal antigen (EEA1), yeast Vac1p, Vps27p, Pip1p in lysosomal protein targeting. Besides the production of D3 phosphorylated lipids, PI3Ks have an intrinsic protein kinase activity. For trimeric GTP-binding protein-activated PI3Kgamma, protein kinase activity seems to be sufficient to trigger mitogen-activated protein kinase (MAPK). Recent disruption of PI3K genes in slime mold, Caenorhabditis elegans, Drosophila melanogaster and mice further underlines the importance of PI3K signaling systems and elucidates the role of PI3K signaling in multicellular organisms.

Signal transmission by many cell surface receptors results in the activation of phosphoinositide (PI) 3-kinases that phosphorylate the 3' position of polyphosphoinositides. From a screen for mouse proteins that bind phosphoinositides, the protein GRP1was identified. GRP1 binds phosphatidylinositol-<em>3,4,5</em>-trisphosphate [<em>PtdIns</em>(3,4, 5)<em>P3</em>] through a pleckstrin homology (PH) domain and displays a region of high sequence similarity to the yeast Sec7 protein. The PH domain of the closely related protein cytohesin-1, which, through its Sec7 homology domain, regulates integrin beta2 and catalyzes guanine nucleotide exchange of the small guanine nucleotide-binding protein ARF1, was also found to specifically bind <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. GRP1 and cytohesin-1 appear to connect receptor-activated PI 3-kinase signaling pathways with proteins that mediate biological responses such as cell adhesion and membrane trafficking.

Pleckstrin homology (PH) domains are protein modules of around 120 amino acids found in many proteins involved in cellular signaling. Certain PH domains drive signal-dependent membrane recruitment of their host proteins by binding strongly and specifically to lipid second messengers produced by agonist-stimulated phosphoinositide 3-kinases (PI 3-Ks). We describe X-ray crystal structures of two different PH domains bound to Ins(1,<em>3,4,5</em>)P4, the head group of the major PI 3-K product <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. One of these PH domains (from Grp1) is <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> specific, while the other (from DAPP1/PHISH) binds strongly to both <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and its 5'-dephosphorylation product, <em>PtdIns</em>(3,4)P2. Comparison of the two structures provides an explanation for the distinct phosphoinositide specificities of the two PH domains and allows us to predict the 3-phosphoinositide selectivity of uncharacterized PH domains.

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol <em>3,4,5</em>-triphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates <em>PtdIns</em>(3,4, 5)<em>P3</em>, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.