3-Phosphoinositide-dependent protein kinase-1 (PDK1) interacts stereoselectively with the d-enantiomer of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> (KD 1.6 nM) and <em>PtdIns</em>(3,4)P2 (KD 5.2 nM), but binds with lower affinity to <em>PtdIns</em>3P or <em>PtdIns</em>(4,5)P2. The binding of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> to PDK1 was greatly decreased by making specific mutations in the pleckstrin homology (PH) domain of PDK1 or by deleting it. The same mutations also greatly decreased the rate at which PDK1 activated protein kinase Balpha (PKBalpha) in vitro in the presence of lipid vesicles containing <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, but did not affect the rate at which PDK1 activated a PKBalpha mutant lacking the PH domain in the absence of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. When overexpressed in 293 or PAE cells, PDK1 was located at the plasma membrane and in the cytosol, but was excluded from the nucleus. Mutations that disrupted the interaction of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> or <em>PtdIns</em>(4,5)P2 with PDK1 abolished the association of PDK1 with the plasma membrane. Growth-factor stimulation promoted the translocation of transfected PKBalpha to the plasma membrane, but had no effect on the subcellular distribution of PDK1 as judged by immunoelectron microscopy of fixed cells. This conclusion was also supported by confocal microscopy of green fluorescent protein-PDK1 in live cells. These results, together with previous observations, indicate that <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> plays several roles in the PDK1-induced activation of PKBalpha. First, it binds to the PH domain of PKB, altering its conformation so that it can be activated by PDK1. Secondly, interaction with <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> recruits PKB to the plasma membrane with which PDK1 is localized constitutively by virtue of its much stronger interaction with <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> or <em>PtdIns</em>(4,5)P2. Thirdly, the interaction of PDK1 with <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> facilitates the rate at which it can activate PKB.

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)P(3)) regulate cell migration, but the role of <em>PtdIns</em>(<em>3,4,5</em>)P(3)-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a <em>PtdIns</em>(<em>3,4,5</em>)P(3) phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the <em>PtdIns</em>(<em>3,4,5</em>)P(3) phosphatase PTEN had no impact on neutrophil chemotaxis. To study <em>PtdIns</em>(<em>3,4,5</em>)P(3) metabolism in living primary cells, we generated a novel transgenic mouse (AktPH-GFP Tg) expressing a bioprobe for <em>PtdIns</em>(<em>3,4,5</em>)P(3.) Time-lapse footage showed rapid, localized binding of AktPH-GFP to the leading edge membrane of chemotaxing ship1(+/+)AktPH-GFP Tg neutrophils, but only diffuse localization in ship1(-/-)AktPH-GFP Tg neutrophils. By directing where <em>PtdIns</em>(<em>3,4,5</em>)P(3) accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.

Polarity is a central feature of eukaryotic cells and phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) has a central role in the polarization of neurons and chemotaxing cells. In polarized epithelial cells, <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> is stably localized at the basolateral plasma membrane, but excluded from the apical plasma membrane, as shown by localization of GFP fused to the <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-binding pleckstrin-homology domain of Akt (GFP-PH-Akt), a fusion protein that indicates the location of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. Here, we ectopically inserted exogenous <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> into the apical plasma membrane of polarized Madin-Darby canine kidney (MDCK) cells. Within 5 min many cells formed protrusions that extended above the apical surface. These protrusions contained basolateral plasma membrane proteins and excluded apical proteins, indicating that their plasma membrane was transformed from apical to basolateral. Addition of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> to the basolateral surface of MDCK cells grown as cysts caused basolateral protrusions. MDCK cells grown in the presence of a phosphatidylinositol 3-kinase inhibitor had abnormally short lateral surfaces, indicating that <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> regulates the formation of the basolateral surface.

Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (<em>PtdIns</em>)-4-P and <em>PtdIns</em>-4,5-P2 and have not considered the lipid products of phosphoinositide 3-kinase: <em>PtdIns</em>-3-P, <em>PtdIns</em>-3,4-P2, and <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to <em>PtdIns</em>-3,4, 5-<em>P3</em> with higher affinity than to <em>PtdIns</em>-4,5-P2, <em>PtdIns</em>-3,4-P2 or inositol 1,<em>3,4,5</em>-tetrakisphosphate (Ins-1,<em>3,4,5</em>-P4). This selectivity is decreased by the xid mutation (R28C). Selective binding of <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> over <em>PtdIns</em>-4,5-P2 or <em>PtdIns</em>-3,4-P2 was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the beta-adrenergic receptor kinase. The oxysterol binding protein and beta-spectrin PH domains bound <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> and <em>PtdIns</em>-4,5-P2 with similar affinities. <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> and <em>PtdIns</em>-4,5-P2 also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin-stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide-3-kinase (PI3K) activity in insulin-stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85alpha regulatory subunit of PI3K (Pik3r1; refs 3-5). Pik3r1-/- mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin-stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full-length p85 alpha in wild-type mice, but via the p50 alpha alternative splicing isoform of the same gene in Pik3r1-/- mice. This isoform switch was associated with an increase in insulin-induced generation of phosphatidylinositol(<em>3,4,5</em>)triphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) in Pik3r1-/- adipocytes and facilitation of Glut4 translocation from the low-density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1-/- mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

The products of PI 3-kinase activation, <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and its immediate breakdown product <em>PtdIns</em>(3,4)P2, trigger physiological processes, by interacting with proteins possessing pleckstrin homology (PH) domains. One of the best characterized <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>/<em>PtdIns</em>(3,4)P2 effector proteins is protein kinase B (PKB), also known as Akt. PKB possesses a PH domain located at its N terminus, and this domain binds specifically to <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and <em>PtdIns</em>(3,4)P2 with similar affinity. Following activation of PI 3-kinase, PKB is recruited to the plasma membrane by virtue of its interaction with <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>/<em>PtdIns</em>(3,4)P2. PKB is then activated by the 3-phosphoinositide-dependent pro-tein kinase-1 (PDK1), which like PKB, possesses a <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>/<em>PtdIns</em>(3,4)P2 binding PH domain. Here, we describe the high-resolution crystal structure of the isolated PH domain of PKB(alpha) in complex with the head group of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. The head group has a significantly different orientation and location compared to other Ins(1,<em>3,4,5</em>)P4 binding PH domains. Mutagenesis of the basic residues that form ionic interactions with the D3 and D4 phosphate groups reduces or abolishes the ability of PKB to interact with <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and <em>PtdIns</em>(3,4)P2. The D5 phosphate faces the solvent and forms no significant interactions with any residue on the PH domain, and this explains why PKB interacts with similar affinity with both <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and <em>PtdIns</em>(3,4)P2.

The effect of phosphoinositides on the activity of protein kinase C (PKC) isotypes was investigated. PKC alpha, beta I, beta II, gamma, delta, epsilon, eta, and zeta were expressed in baculovirus-infected insect cells and purified by column chromatography. The calcium-activated PKC isotypes alpha, beta I, beta II, and gamma were not significantly activated by any of the phosphoinositides investigated (phosphatidylinositol-4-phosphate (<em>PtdIns</em>-4-P), <em>PtdIns</em>-3-P, <em>PtdIns</em>-4,5-P2, <em>PtdIns</em>-3,4-P2, and <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>) when added in the presence of concentrations of phosphatidylserine that give maximal stimulation. The calcium-insensitive PKC isotypes delta, epsilon, and theta also showed little response to <em>PtdIns</em>-3-P, <em>PtdIns</em>-4-P, or <em>PtdIns</em>-4,5-P2 when these lipids were added in the presence of phosphatidylserine. In contrast, <em>PtdIns</em>-3,4-P2 and <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> caused a 5-15-fold stimulation of these enzymes compared with phosphatidylserine alone. 50% maximal stimulation of PKC epsilon by <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> occurred when this lipid was present at about 1% of the carrier <em>PtdIns</em>-4,5-P2 (about 100 nM). These lipids had little effect on baculovirus-expressed PKC zeta, which was constitutively active. A short chain version of <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>, dioctanoyl-<em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>, activated PKC delta, epsilon, and eta in the absence of other lipids, whereas a short chain version of <em>PtdIns</em>-4,5-P2, dihexanoyl-<em>PtdIns</em>-4,5-P2, did not. Since <em>PtdIns</em>-3,4-P2 and <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> are nominally absent in unstimulated cells and appear within seconds to minutes of stimulation by various cell activators, these lipids could act as second messengers to activate PKC delta, epsilon, or eta in vivo.

Although multiple members of the phosphatidylinositol-3-kinase pathway (PI3K) are targeted by germline or somatic mutations, functional mutations in the three akt isoforms have proven elusive. This is somewhat surprising, as AKT represents a key node in the PI3K pathway, exhibiting transforming activity when incorporated into the AKT8 retrovirus. A recent report in Nature identifies a transforming E17K PH domain mutation in akt1 in breast (8%), colorectal (6%), and ovarian (2%) cancers. E17K-akt1 transforming activity appears due to <em>PtdIns</em>(3,4)P2- and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-independent recruitment of AKT1 to the membrane. This novel observation raises important theoretical and clinical questions.

Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating <em>PtdIns</em>(3,4)P2 and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind <em>PtdIns</em>(3,4)P2 and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind <em>PtdIns</em>(3,4)P2 (AKT) or <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to <em>PtdIns</em>(3,4)P2 and/or <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to <em>PtdIns</em>(3, 4)P2 and/or <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.

The production, survival, and function of monocytes and macrophages is regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms. Binding of M-CSF to Fms induces the tyrosine phosphorylation and association of a 150-kD protein with the phosphotyrosine-binding (PTB) domain of Shc. We have cloned p150 using a modified yeast two-hybrid screen. p150 contains one SH2 domain, two potential PTB-binding sites, an ATP/GTP-binding domain, several potential SH3-binding sites, and a domain with homology to inositol polyphosphate-5-phosphatases. p150 antibodies detect this protein in FDC-P1 myeloid cells, but the same protein is not detectable in fibroblasts. The antibodies immunoprecipitate a 150-kD protein from quiescent or M-CSF-stimulated FDC-P1 cells that hydrolyzes <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, to <em>PtdIns</em>(3,4)P2. This activity is observed in Shc immunoprecipitates only after M-CSF stimulation. Retroviral expression of p15O in FD-Fms cells results in strong inhibition of cell growth in M-CSF and a lesser inhibition in IL-3. Ectopic expression of p150 in fibroblasts does not inhibit growth. This novel protein, p150(ship) (SH2-containing inositol phosphatase), identifies a component of a new growth factor-receptor signaling pathway in hematopoietic cells.

Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-<em>3,4,5</em>-trisphosphate (<em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cgamma (PLCgamma) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> regulates receptor-dependent calcium signals through the function of Tec kinases.

Recent evidence has suggested that activation of phosphoinositide 3-kinase (PI 3-kinase) is required for the activation of Akt-1 by growth factors and insulin. Here we demonstrate by two independent methods that Akt-1 from L6 myotubes binds to <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, <em>PtdIns</em>(3,4)P2 and <em>PtdIns</em>(4,5)P2 when presented against a background of phosphatidylserine (PtdSer) or a 1:1 mixture of PtdSer and phosphatidylcholine (PtdCho). No binding was observed with the lipids <em>PtdIns</em>(3,5)P2, <em>PtdIns</em>4P and <em>PtdIns</em>3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> in a similar manner as inactive Akt-1. Quantitative analysis using surface plasmon resonance showed that the equilibrium association constant for the binding of Akt-1 to <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> was submicromolar and that <em>PtdIns</em>(3,4)P2 and <em>PtdIns</em>(4,5)P2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.

The aberrant activity of Ras homologous (Rho) family small GTPases (20 human members) has been implicated in cancer and other human diseases. However, in contrast to the direct mutational activation of Ras found in cancer and developmental disorders, Rho GTPases are activated most commonly in disease by indirect mechanisms. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs). RhoGEFs promote formation of the active GTP-bound state of Rho GTPases. The largest family of RhoGEFs is comprised of the Dbl family RhoGEFs with 70 human members. The multitude of RhoGEFs that activate a single Rho GTPase reflects the very specific role of each RhoGEF in controlling distinct signaling mechanisms involved in Rho activation. In this review, we summarize the role of Dbl RhoGEFs in development and disease, with a focus on Ect2 (epithelial cell transforming squence 2), Tiam1 (T-cell lymphoma invasion and metastasis 1), Vav and P-Rex1/2 (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> (phosphatidylinositol (<em>3,4,5</em>)-triphosphate)-dependent Rac exchanger).

BACKGROUND

Protein kinase B (PKB) is activated by phosphorylation of Thr308 and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is unknown.

RESULTS

The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF). PIF is situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBalpha, except that the residue equivalent to Ser473 is aspartic acid. Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF with PDK1. Remarkably, interaction of PDK1 with PIF, or with a synthetic peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBalpha to one that phosphorylates both Thr308 and Ser473 of PKBalpha in a manner dependent on phosphatidylinositol (<em>3,4,5</em>) trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>). Furthermore, the interaction of PIF with PDK1 converted the PDK1 from a form that is not directly activated by <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> to a form that is activated threefold by <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. We have partially purified a kinase from brain extract that phosphorylates Ser473 of PKBalpha in a <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-dependent manner and that is immunoprecipitated with PDK1 antibodies.

CONCLUSIONS

PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1.

Chemoattractant-responsive cells are able to translate a shallow extracellular chemical gradient into a steep intracellular gradient resulting in the localization of F-actin assembly at the front and an actomyosin network at the rear that moves the cell forward. Recent evidence suggests that one of the first asymmetric cellular responses is the localized accumulation of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, the product of class I phosphoinositide 3-kinase (PI3K) at the site of the new leading edge. The strong accumulation of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> results from the localized activation of PI3K and also from feedback loops that amplify <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> synthesis at the front and control its degradation at the side and back of cells. These different pathways are temporally and spatially regulated and integrate with other signaling pathways during directional sensing and chemotaxis.

Protein kinase B (PKB/Akt) is a key regulator of cell growth, proliferation and metabolism. It possesses an N-terminal pleckstrin homology (PH) domain that interacts with equal affinity with the second messengers <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and <em>PtdIns</em>(3,4)P2, generated through insulin and growth factor-mediated activation of phosphoinositide 3-kinase (PI3K). The binding of PKB to <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>/<em>PtdIns</em>(3,4)P2 recruits PKB from the cytosol to the plasma membrane and is also thought to induce a conformational change that converts PKB into a substrate that can be activated by the phosphoinositide-dependent kinase 1 (PDK1). In this study we describe two high-resolution crystal structures of the PH domain of PKBalpha in a noncomplexed form and compare this to a new atomic resolution (0.98 A, where 1 A=0.1 nm) structure of the PH domain of PKBalpha complexed to Ins(1,<em>3,4,5</em>)P4, the head group of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. Remarkably, in contrast to all other PH domains crystallized so far, our data suggest that binding of Ins(1,<em>3,4,5</em>)P4 to the PH domain of PKB, induces a large conformational change. This is characterized by marked changes in certain residues making up the phosphoinositide-binding site, formation of a short a-helix in variable loop 2, and a movement of variable loop 3 away from the lipid-binding site. Solution studies with CD also provided evidence of conformational changes taking place upon binding of Ins(1,<em>3,4,5</em>)P4 to the PH domain of PKB. Our data provides the first structural insight into the mechanism by which the interaction of PKB with <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>/<em>PtdIns</em>(3,4)P2 induces conformational changes that could enable PKB to be activated by PDK1.

BACKGROUND

Protein kinase B (PKB) is involved in the regulation of apoptosis, protein synthesis and glycogen metabolism in mammalian cells. Phosphoinositide-dependent protein kinase (PDK-1) activates PKB in a manner dependent on phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>), which is also needed for the translocation of PKB to the plasma membrane. It has been proposed that the amount of PKB activated is determined exclusively as a result of its translocation, and that a constitutively active pool of membrane-associated PDK-1 simply phosphorylates all the PKB made available. Here, we have investigated the effects of membrane localisation of PDK-1 on PKB activation.

RESULTS

Ectopically expressed PDK-1 translocated to the plasma membrane in response to platelet-derived growth factor (PDGF) and translocation was sensitive to wortmannin, an inhibitor of phosphoinositide 3-kinase. Translocation of PDK-1 also occurred upon its co-expression with constitutively active phosphoinositide 3-kinase, but not with an inactive form. Overexpression of PDK-1 enhanced the ability of PDGF to activate PKB. PDK-1 disrupted in the pleckstrin homology (PH) domain which did not translocate to the membrane did not increase PKB activity in response to PDGF, whereas membrane-targeted PDK-1 activated PKB to the extent that it could not be activated further by PDGF.

CONCLUSIONS

In response to PDGF, binding of Ptdlns (<em>3,4,5</em>)<em>P3</em> and/or Ptdlns(3,4)P2 to the PH domain of PDK-1 causes its translocation to the plasma membrane where it co-localises with PKB, significantly contributing to the scale of PKB activation.

The evolutionarily conserved DOCK180 protein has an indispensable role in cell migration by functioning as an exchange factor for Rac GTPase via its DOCK homology region (DHR)-2 domain. We report here that the conserved DHR-1 domain also has an important signalling role. A form of DOCK180 that lacks DHR-1 fails to promote cell migration, although it is capable of inducing Rac GTP-loading. The DHR-1 domain interacts with <em>PtdIns</em>(<em>3,4,5</em>)P(3) in vitro and in vivo, and mediates the DOCK180 signalling complex localization at sites of <em>PtdIns</em>(<em>3,4,5</em>)P(3) accumulation in the cell's leading edge. A form of DOCK180 in which the DHR-1 domain has been replaced by a canonical <em>PtdIns</em>(<em>3,4,5</em>)P(3)-binding pleckstrin homology domain is fully functional at inducing cell elongation and migration, suggesting that the main function of DHR-1 is to bind <em>PtdIns</em>(<em>3,4,5</em>)P(3). These results demonstrate that DOCK180, via its DHR-1 and DHR-2 domains, couples <em>PtdIns</em>(<em>3,4,5</em>)P(3) signalling to Rac GTP-loading, which is essential for directional cell movement.

Inositol phosphates are well-known signaling molecules, whereas the inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP7/IP7) and bis-diphosphoinositol tetrakisphosphate (InsP8/IP8), are less well characterized. We demonstrate physiologic regulation of Dictyostelium chemotaxis by InsP7 mediated by its competition with <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> for binding pleckstrin homology (PH) domain-containing proteins. Chemoattractant stimulation triggers rapid and sustained elevations in InsP7/InsP8 levels. Depletion of InsP7 and InsP8 by deleting the gene for InsP6 kinase (InsP6K/IP6K), which converts inositol hexakisphosphate (InsP6/IP6) to InsP7, causes rapid aggregation of mutant cells and increased sensitivity to cAMP. Chemotaxis is mediated by membrane translocation of certain PH domain-containing proteins via specific binding to <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. InsP7 competes for PH domain binding with <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> both in vitro and in vivo. InsP7 depletion enhances PH domain membrane translocation and augments downstream chemotactic signaling activity.

The tumor suppressor gene PTEN, which is frequently mutated in human cancers, encodes a lipid phosphatase for phosphatidylinositol <em>3,4,5</em>-triphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>] and antagonizes phosphatidylinositol 3 kinase. Primordial germ cells (PGCs), which are the embryonic precursors of gametes, are the source of testicular teratoma. To elucidate the intracellular signaling mechanisms that underlie germ cell differentiation and proliferation, we have generated mice with a PGC-specific deletion of the Pten gene. Male mice that lacked PTEN exhibited bilateral testicular teratoma, which resulted from impaired mitotic arrest and outgrowth of cells with immature characters. Experiments with PTEN-null PGCs in culture revealed that these cells had greater proliferative capacity and enhanced pluripotent embryonic germ (EG) cell colony formation. PTEN appears to be essential for germ cell differentiation and an important factor in testicular germ cell tumor formation.

The regulatory and catalytic properties of the three mammalian isoforms of protein kinase B (PKB) have been compared. All three isoforms (PKBalpha, PKBbeta and PKBgamma) were phosphorylated at similar rates and activated to similar extents by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Phosphorylation and activation of each enzyme required the presence of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> or <em>PtdIns</em>(3,4)P2, as well as PDK1. The activation of PKBbeta and PKBgamma by PDK1 was accompanied by the phosphorylation of the residues equivalent to Thr308 in PKBalpha, namely Thr309 (PKBbeta) and Thr305 (PKBgamma). PKBgamma which had been activated by PDK1 possessed a substrate specificity identical with that of PKBalpha and PKBbeta towards a range of peptides. The activation of PKBgamma and its phosphorylation at Thr305 was triggered by insulin-like growth factor-1 in 293 cells. Stimulation of rat adipocytes or rat hepatocytes with insulin induced the activation of PKBalpha and PKBbeta with similar kinetics. After stimulation of adipocytes, the activity of PKBbeta was twice that of PKBalpha, but in hepatocytes PKBalpha activity was four-fold higher than PKBbeta. Insulin induced the activation of PKBalpha in rat skeletal muscle in vivo, with little activation of PKBbeta. Insulin did not induce PKBgamma activity in adipocytes, hepatocytes or skeletal muscle, but PKBgamma was the major isoform activated by insulin in rat L6 myotubes (a skeletal-muscle cell line).

The diverse effects mediated by PI3K/PTEN (phosphoinositide 3-kinase/phosphatase and tensin homologue deleted on chromosome 10) signalling in the heart clearly support an important biological and pathophysiological role for this signalling cascade. PI3Ks are a family of evolutionarily conserved lipid kinases that mediate many cellular responses to physiological and pathophysiological stimuli. Class I PI3K can be activated by either receptor tyrosine kinase/cytokine receptor activation (class IA) or G-protein-coupled receptors (class IB), leading to the generation of phosphatidyl inositol (<em>3,4,5</em>)<em>P3</em> and recruitment and activation of Akt/protein kinase B, 3'-phosphoinositide-dependent kinase-1 (PDK1), or monomeric G-proteins, and phosphorylation of a wide range of downstream targets including glycogen synthase kinase 3beta (GSK3beta), mTOR (mammalian target of rapamycin), p70S6 kinase, endothelial nitric oxide synthase, and several anti-apoptotic effectors. Class IA (PI3Kalpha, beta, and delta) and class IB (PI3Kgamma) PI3Ks mediate distinct phenotypes in the heart under negative control by the 3'-lipid phosphatase PTEN, which dephosphorylates <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> to generate <em>PtdIns</em>(4,5)P2. PI3Kalpha, PI3Kgamma, and PTEN are expressed in cardiomyocytes, fibroblasts, endothelial cells, and vascular smooth muscle cells, where they modulate cell survival, hypertrophy, contractility, metabolism, and mechanotransduction. The PI3K/PTEN signalling pathways are involved in a wide variety of diseases including myocardial hypertrophy and contractility, heart failure, and preconditioning. In this review, we discuss the signalling pathways mediated by PI3K class I isoforms and PTEN and their roles in cardiac structure and function.

PTEN dysfunction plays a crucial role in the pathogenesis of hereditary and sporadic cancers. Here, we show that PTEN homodimerizes and, in this active conformation, exerts lipid phosphatase activity on <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. We demonstrate that catalytically inactive cancer-associated PTEN mutants heterodimerize with wild-type PTEN and constrain its phosphatase activity in a dominant-negative manner. To study the consequences of homo- and heterodimerization of wild-type and mutant PTEN in vivo, we generated Pten knockin mice harboring two cancer-associated PTEN mutations (PtenC124S and PtenG129E). Heterozygous Pten(C124S/+) and Pten(G129E/+) cells and tissues exhibit increased sensitivity to PI3-K/Akt activation compared to wild-type and Pten(+/-) counterparts, whereas this difference is no longer apparent between Pten(C124S/-) and Pten(-/-) cells. Notably, Pten KI mice are more tumor prone and display features reminiscent of complete Pten loss. Our findings reveal that PTEN loss and PTEN mutations are not synonymous and define a working model for the function and regulation of PTEN.

Polarized cell movement is triggered by the development of a <em>PtdIns</em>(<em>3,4,5</em>)P(3) gradient at the membrane, which is followed by rearrangement of the actin cytoskeleton. The WASP family verprolin homologous protein (WAVE) is essential for lamellipodium formation at the leading edge by activating the Arp2/3 complex downstream of Rac GTPase. Here, we report that WAVE2 binds to <em>PtdIns</em>(<em>3,4,5</em>)P(3) through its basic domain. The amino-terminal portion of WAVE2, which includes the <em>PtdIns</em>(<em>3,4,5</em>)P(3)-binding sequence, was localized at the leading edge of lamellipodia induced by an active form of Rac (RacDA) or by treatment with platelet-derived growth factor (PDGF). Production of <em>PtdIns</em>(<em>3,4,5</em>)P(3) at the cell membrane by myristoylated phosphatidylinositol-3-OH kinase (PI(3)K) is sufficient to recruit WAVE2 in the presence of dominant-negative Rac and latrunculin, demonstrating that <em>PtdIns</em>(<em>3,4,5</em>)P(3) alone is able to recruit WAVE2. Expression of a full-length mutant of WAVE2 that lacks the lipid-binding activity inhibited proper formation of lamellipodia induced by RacDA. These results suggest that one of the products of PI(3)K, <em>PtdIns</em>(<em>3,4,5</em>)P(3), recruits WAVE2 to the polarized membrane and that this recruitment is essential for lamellipodium formation at the leading edge.