We used high-resolution mass spectrometry to map the cytotoxic T lymphocyte (CTL) proteome and the effect of the metabolic checkpoint kinase mTORC1 on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes, and mTORC1 selectively repressed and promoted expression of a subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for negative control of the production of phosphatidylinositol (<em>3,4,5</em>)-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) by mTORC1 in CTLs. mTORC1 repressed <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> production and determined the requirement for mTORC2 in activation of the kinase Akt. Our unbiased proteomic analysis thus provides comprehensive understanding of CTL identity and the control of CTL function by mTORC1.

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol <em>3,4,5</em>-trisphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>] to polarized MDCK cells was sufficient to increase PAK internalization. <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.

Phosphatidylinositol 3,4-bisphosphate (<em>PtdIns</em>(3,4)P2) and phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) are lipid second messengers that regulate various cellular processes by recruiting a wide range of downstream effector proteins to membranes. Several pleckstrin homology (PH) domains have been reported to interact with <em>PtdIns</em>(3,4)P2 and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. To understand how these PH domains differentially respond to <em>PtdIns</em>(3,4)P2 and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> signals, we quantitatively determined the <em>PtdIns</em>(3,4)P2 and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> binding properties of several PH domains, including Akt, ARNO, Btk, DAPP1, Grp1, and C-terminal TAPP1 PH domains by surface plasmon resonance and monolayer penetration analyses. The measurements revealed that these PH domains have significant different phosphoinositide specificities and affinities. Btk-PH and TAPP1-PH showed genuine <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and <em>PtdIns</em>(3,4)P2 specificities, respectively, whereas other PH domains exhibited less pronounced specificities. Also, the PH domains showed different degrees of membrane penetration, which greatly affected the kinetics of their membrane dissociation. Mutational studies showed that the presence of two proximal hydrophobic residues on the membrane-binding surface of the PH domain is important for membrane penetration and sustained membrane residence. When NIH 3T3 cells were stimulated with platelet-derived growth factor to generate <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, reversible translocation of Btk-PH, Grp1-PH, ARNO-PH, DAPP1-PH, and its L177A mutant to the plasma membrane was consistent with their in vitro membrane binding properties. Collectively, these studies provide new insight into how various PH domains would differentially respond to cellular <em>PtdIns</em>(3,4)P2 and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> signals.

Phagocytosis and chemotaxis are receptor-mediated processes that require extensive rearrangements of the actin cytoskeleton, and are controlled by lipid second messengers such as phosphatidylinositol <em>3,4,5</em>-trisphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>] and phosphatidylinositol 3,4-bisphosphate [<em>PtdIns</em>(3,4)P2]. We used a panel of pleckstrin homology (PH) domains with distinct binding specificities for <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and <em>PtdIns</em>(3,4)P2 to study the spatiotemporal dynamics of these phosphoinositides in vivo. During phagocytosis and macropinocytosis <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> levels transiently increased at sites of engulfment, followed by a rapid <em>PtdIns</em>(3,4)P2 production round the phagosome/macropinosome upon its internalisation, suggesting that <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> is degraded to <em>PtdIns</em>(3,4)P2. PTEN null mutants, which are defective in phagocytosis, showed normal rates of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> degradation, but unexpectedly an accelerated <em>PtdIns</em>(3,4)P2 degradation. During chemotaxis to cAMP only <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> was formed in the plasma membrane, and no <em>PtdIns</em>(3,4)P2 was detectable, showing that all <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> was degraded by PTEN to <em>PtdIns</em>(4,5)P2. Furthermore, we showed that different <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> binding PH domains gave distinct spatial and temporal readouts of the same underlying <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> signal, enabling distinct biological responses to one signal.

Phosphatidylinositol (<em>3,4,5</em>) trisphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>] is a lipid second messenger, produced by Type I phosphoinositide 3-kinases (PI 3-kinases), which mediates intracellular responses to many growth factors. Although PI 3-kinases are implicated in events at both the plasma membrane and intracellular sites, including the nucleus, direct evidence for the occurrence of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> at non-plasma membrane locations is limited. We made use of the pleckstrin homology (PH) domain of general receptor for phosphoinositides (Grp1) to detect <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> in an on-section labeling approach by quantitative immunogold electron microscopy. Swiss 3T3 cells contained low levels of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> that increased up to 15-fold upon stimulation with platelet-derived growth factor (PDGF). The signal was sensitive to PI 3-kinase inhibitors and present mainly at plasma membranes, including lamellipodia, and in a surprisingly large pool within the nuclear matrix. Comparatively little labeling was observed in endomembranes. A similar distribution of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> was observed in U87MG cells, which lack the <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> phosphatase, PTEN. Re-expression of PTEN into U87MG cells ablated plasma membrane <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, but not the nuclear pool of this lipid even when PTEN was targeted to nuclei. These data have important implications for the versatility of PI 3-kinase signaling and for the proposed functions of PTEN in the nucleus.

It has been demonstrated that the lipid products of the phosphoinositide 3-kinase (PI3K) can associate with the Src homology 2 (SH2) domains of specific signaling molecules and modify their actions. In the current experiments, phosphatidylinositol 3,4, 5-trisphosphate (<em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>) was found to bind to the C-terminal SH2 domain of phospholipase Cgamma (PLCgamma) with an apparent Kd of 2.4 microM and to displace the C-terminal SH2 domain from the activated platelet-derived growth factor receptor (PDGFR). To investigate the in vivo relevance of this observation, intracellular inositol trisphosphate (I<em>P3</em>) generation and calcium release were examined in HepG2 cells expressing a series of PDGFR mutants that activate PLCgamma with or without receptor association with PI3K. Coactivation of PLCgamma and PI3K resulted in an approximately 40% increase in both intracellular I<em>P3</em> generation and intracellular calcium release as compared with selective activation of PLCgamma. Similarly, the addition of wortmannin or LY294002 to cells expressing the wild-type PDGFR inhibited the release of intracellular calcium. Thus, generation of <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> by receptor-associated PI3K causes an increase in I<em>P3</em> production and intracellular calcium release, potentially via enhanced <em>PtdIns</em>-4, 5-P2 substrate availability due to <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>-mediated recruitment of PLCgamma to the lipid bilayer.

Cellular levels of phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) are rapidly elevated in response to activation of growth factor receptor tyrosine kinases. This polyphosphoinositide binds the pleckstrin homology (PH) domain of GRP1, a protein that also contains 200 residues with high sequence similarity to a segment of the yeast Sec7 protein that functions as an ADP ribosylation exchange factor (ARF) (Klarlund, J., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that dioctanoyl <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> binds the PH domain of GRP1 with a Kd = 0.5 microM, an affinity 2 orders of magnitude greater than dioctanoyl-<em>PtdIns</em>(4,5)P2. Further, the Sec7 domain of GRP1 is found to catalyze guanine nucleotide exchange of ARF1 and -5 but not ARF6. Importantly, <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, but not <em>PtdIns</em>(4,5)P2, markedly enhances the ARF exchange activity of GRP1 in a reaction mixture containing dimyristoylphosphatidylcholine micelles, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and a low concentration of sodium cholate. <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-mediated ARF nucleotide exchange through GRP1 is selectively blocked by 100 microM inositol 1,<em>3,4,5</em>-tetrakisphosphate, which also binds the PH domain of GRP1. Taken together, these data are consistent with the hypothesis that selective recruitment of GRP1 to <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> in membranes activates ARF1 and -5, known regulators of intracellular membrane trafficking.

BACKGROUND

Shc and Grb2 form a complex in cells in response to growth factor stimulation and link tyrosine kinases to Ras during the resulting signaling process. Shc and Grb2 each contain domains that mediate interactions with other unidentified intracellular proteins. For example, the Shc PTB domain binds to 130 kDa and 145 kDa tyrosine-phosphorylated proteins in response to stimulation of cells by growth factors, cytokines and crosslinking of antigen receptors. The Grb2 SH3 domains bind to an unidentified 116 kDa protein in T cells. We have identified three proteins, of 110 kDa, 130 kDa and 145 kDa, as a new family of molecules encoded by the same gene. In vivo studies show that these proteins form signal transduction complexes with Shc and with Grb2.

RESULTS

The 130 kDa and 145 kDa tyrosine-phosphorylated proteins that associate with the Shc PTB domain were purified by conventional chromatographic methods. Partial peptide and cDNA sequences corresponding to these proteins, termed SIP-145 and SIP-130 (SIP for signaling inositol polyphosphate 5-phosphatase), identified them as SH2 domain-containing products of a single gene and as members of the inositol polyphosphate 5-phosphatase family. The SIP-130 and SIP-145 proteins and inositol polyphosphate 5-phosphatase activity associated with Shc in vivo in response to B-cell activation. By using an independent approach, expression cloning, we found that the Grb2 SH3 domains bind specifically to SIP-110, a 110 kDa splice variant of SIP-145 and SIP-130, which lacks the SH2 domain. The SIP proteins hydrolyzed phosphatidylinositol (<em>3,4,5</em>)-trisphosphate (<em>PtdIns</em> (<em>3,4,5</em>)-<em>P3</em>) and Ins (1,<em>3,4,5</em>)-P4, but not <em>PtdIns</em> (4,5)-P2 or Ins (1,4,5)-<em>P3</em>.

CONCLUSIONS

These findings strongly implicate the inositol polyphosphate 5-phosphatases in Shc- and Grb2-mediated signal transduction. Furthermore, SIP-110, SIP-130 and SIP-145 prefer 3-phosphorylated substrates, suggesting a link to the phosphatidylinositol 3-kinase signaling pathway.

Lipid second messengers, particularly those derived from the polyphosphoinositide metabolism, play a pivotal role in multiple cell signaling networks. Phosphoinositide 3-kinase (PI3K) generate 3'-phosphorylated inositol lipids that are key players in a multitude of cell functions. One of the best characterized targets of PI3K lipid products is the serine/threonine protein kinase Akt (protein kinase B, PKB). Recent findings have implicated the PI3K/Akt pathway in tumorigenesis because it stimulates cell proliferation and suppresses apoptosis. However, it was thought that this signal transduction network would exert its carcinogenetic effects mainly by operating in the cytoplasm. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid cycle, and strongly suggests that lipid molecules are important components of signaling pathways operating at the nuclear level. PI3K, its lipid product phosphatidylinositol (<em>3,4,5</em>) trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>), and Akt have been identified within the nucleus and recent data suggest that they counteract apoptosis also by operating in this cell compartment through a block of caspase-activated DNase and inhibition of chromatin condensation. In this review, we shall summarize the most updated and intriguing findings about nuclear PI3K/<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>/Akt in relationship with tumorigenesis and suppression of apoptotic stimuli.

The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [<em>PtdIns</em>(3,4)P2] and phosphatidylinositol <em>3,4,5</em>-triphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both <em>PtdIns</em>(3, 4)P2 and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, whereas <em>PtdIns</em>(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G(1) in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27(KIP1). Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

Phosphoinositide 30H-kinase (PI3K) activities are thought to be critical regulatory enzymes in a new intracellular signalling pathway, the activation of which results in the rapid accumulation of a putative signalling molecule, phosphatidylinositol (<em>3,4,5</em>)-trisphosphate [<em>PtdIns</em>(<em>3,4,5</em>) <em>P3</em>]. To date, activation of PI3K has always correlated with its recruitment into complexes containing protein tyrosine kinases (PTK). Here we report that agonists which utilize G-protein mediated transduction pathways can stimulate very rapid and large accumulations of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> via a novel mechanism, possibly involving direct coupling between the G-protein and a PI3K activity. In addition, some of these agonists also stimulate small increases in PI3K activity in anti-phosphotyrosine and anti-src-type PTK antibody directed immunoprecipitates, indicating activation of PI3K via a 'conventional' PTK mediated mechanism; these pathways however, play only a minor role in the initial, agonist sensitive production of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> in myeloid derived cells.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (<em>3,4,5</em>)-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, which implies that the PH domain confers <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.

PTEN is a tumor suppressor gene that is frequently mutated in human tumors. It functions primarily as a lipid phosphatase and plays a key role in the regulation of phosphatidylinositol-3'-kinase. PTEN appears to play a crucial role in modulating apoptosis by reducing the levels of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, a phospholipid that activates AKT, a central regulator of apoptosis. To understand the role of PTEN in regulating cell proliferation and apoptosis, we stably overexpressed PTEN in PC3 cells, which are prostate cancer cells that lack PTEN. Overexpression of PTEN in two different clones inhibited cell proliferation and increased serum starvation-induced apoptosis, as compared to control cells. Interestingly, PTEN overexpression resulted in a 44-60% reduction in total insulin-like growth factor-I receptor (IGF-IR) protein levels and a 49-64% reduction in cell surface IGF-IR expression. [35S]methionine pulse experiments in PC3 cells overexpressing PTEN demonstrated that these cells synthesize significantly lower levels of the IGF-IR precursor, whereas PTEN overexpression had no effect on IGF-IR degradation. Taken together, our results show that PTEN can regulate cell proliferation and apoptosis through inhibition of IGF-IR synthesis. These results have important implications for understanding the roles of PTEN and the IGF-IR in prostate cancer cell tumorigenesis.

In obesity and diabetes, the ability of hypothalamic neurons to sense and transduce changes in leptin and insulin levels is compromised. The effects of both hormones require intracellular signalling via the PI3-kinase pathway, which is inhibited by the phosphatase PTEN. We show that leptin-stimulated F-actin depolymerization in mouse hypothalamic cells is inhibited by PTEN, a process involving independent effects of both its lipid and protein phosphatase activities. Potentially mediating this F-actin depolymerization, leptin, but not insulin, stimulated the phosphorylation of PTEN in a CK2 dependent manner, and inhibited its phosphatase activity. Similarly, hyperpolarization of mouse pancreatic beta-cells by leptin also requires coincident <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> generation and actin depolymerization, and could be inhibited by mechanisms requiring both the lipid and protein phosphatase activities of PTEN. These results demonstrate a critical role for PTEN in leptin signalling and indicate a mechanism by which leptin and insulin can produce PI3K dependent differential cellular outputs.

Insulin-stimulated glucose uptake involves the recruitment of the glucose transporter 4 isoform (GLUT4) from an intracellular location to the plasma membrane of fat and muscle cells. Although the activation of the PI3-kinase/protein kinase B (PKB) pathway is central to this effect of insulin, the key substrates for PKB that are involved require identification. Here we report that serine318 on the FYVE domain-containing <em>PtdIns</em>3P 5-kinase (PIKfyve) is a novel substrate for PKB, and show that phosphorylation stimulates the <em>PtdIns</em>3P 5-kinase activity of the enzyme. We also demonstrate that PIKfyve is phosphorylated on serine318 in intact cells in response to insulin, in a PI3-kinase-dependent manner, and that PIKfyve colocalises with a highly motile subpopulation of insulin-regulated aminopeptidase (IRAP)/GLUT4 vesicles. Finally, we demonstrate that overexpression of a PIKfyve[S318A] mutant in 3T3-L1 adipocytes enhances insulin-stimulated IRAP/GLUT4 vesicle translocation to the plasma membrane suggesting a role for PKB-dependent phosphorylation of PIKfyve in insulin-regulated IRAP/GLUT4 trafficking. The phosphorylation and activation of PIKfyve by PKB provides a novel signalling paradigm that may link plasma membrane-localised <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> signals via a protein kinase cascade to regulated <em>PtdIns</em>(3,5)P2 production, and thereby to the control of trafficking of other membrane cargos.

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (<em>PtdIns</em>(3,4)P2) but not of <em>PtdIns</em>(<em>3,4,5</em>,)<em>P3</em> is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (<em>PtdIns</em> 3-kinase) and is accompanied by a significant increase in <em>PtdIns</em> 3-kinase activity in this subcellular fraction. Actually, <em>PtdIns</em>(3,4)P2 accumulation and <em>PtdIns</em> 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of <em>PtdIns</em>(3,4)P2 and the relocalization of <em>PtdIns</em> 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with <em>PtdIns</em> 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that <em>PtdIns</em> 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for <em>PtdIns</em> 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>]. In order to study the effects of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8% identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, inositol 1, <em>3,4,5</em>-tetrakisphosphate [Ins(1,<em>3,4,5</em>)P4], with high affinity (Kd 32. 2+/-5.2 nM) and inositol phosphate specificity [Kd values for Ins(1, <em>3,4,5</em>,6)P5, InsP6, Ins(1,3,4)<em>P3</em> and Ins(1,4,5)<em>P3</em>: 283+/-32, >10000, >10000 and >10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,<em>3,4,5</em>)P4, data that are consistent with its proposed role as a putative <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> receptor. To address whether GRP1 binds <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP-GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and hence constitutes a true <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> receptor.

Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (<em>PtdIns</em>(4,5)P(2)), and phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)P(3)) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca(2+)-dependent C2 domain of protein kinase Calpha (PKCalpha) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCalpha-C2 nonspecifically binds phosphoinositides, including <em>PtdIns</em>(4,5)P(2) and <em>PtdIns</em>(<em>3,4,5</em>)P(3), but that PS and Ca(2+) binding is prerequisite for productive phosphoinositide binding. <em>PtdIns</em>(4,5)P(2) or <em>PtdIns</em>(<em>3,4,5</em>)P(3) augments the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca(2+)-dependent PS binding is essential for membrane interactions of PKCalpha-C2. <em>PtdIns</em>(4,5)P(2) alone cannot drive the membrane attachment of the domain but further stabilizes the Ca(2+)- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCalpha-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of <em>PtdIns</em>(4,5)P(2) and/or <em>PtdIns</em>(<em>3,4,5</em>)P(3) from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of <em>PtdIns</em>(4,5)P(2) or <em>PtdIns</em>(<em>3,4,5</em>)P(3) at the PM slowed membrane dissociation of PKCalpha-C2. Collectively, these studies show that <em>PtdIns</em>(4,5)P(2) and <em>PtdIns</em>(<em>3,4,5</em>)P(3) augment the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCalpha-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.

SHIP-2 is a phosphoinositidylinositol <em>3,4,5</em> trisphosphate (<em>PtdIns</em>[<em>3,4,5</em>]<em>P3</em>) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline-rich domain. To identify proteins that bind to the SHIP-2 proline-rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

Many neutrophil functions are regulated by phosphatidylinositol-<em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) that mediates protein membrane translocation via binding to pleckstrin homolog (PH) domains within target proteins. Here we show that inositol 1,<em>3,4,5</em>-tetrakisphosphate (Ins(1,<em>3,4,5</em>)P4), a cytosolic small molecule, bound the same PH domain of target proteins and competed for binding to <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. In neutrophils, chemoattractant stimulation triggered rapid elevation in Ins(1,<em>3,4,5</em>)P4 concentration. Depletion of Ins(1,<em>3,4,5</em>)P4 by deleting the gene encoding Ins<em>P3</em>KB, which converts Ins(1,4,5)<em>P3</em> to Ins(1,<em>3,4,5</em>)P4, enhanced membrane translocation of the <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-specific PH domain. This led to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. On the contrary, augmentation of intracellular Ins(1,<em>3,4,5</em>)P4 concentration blocked PH domain-mediated membrane translocation of target proteins and dramatically decreased the sensitivity of neutrophils to chemoattractant stimulation. These findings establish a role for Ins(1,<em>3,4,5</em>)P4 in cellular signal transduction pathways and provide another mechanism for modulating <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> signaling in neutrophils.

As potential targets for polyphosphoinositides, activation of protein kinase C (PKC) isotypes (beta 1, epsilon, zeta, nu) and a member of the PKC-related kinase (PRK) family, PRK1, has been compared in vitro. PRK1 is shown to be activated by both phosphatidylinositol 4,5-bisphosphate (<em>PtdIns</em> 4,5-P2) as well as phosphatidylinositol <em>3,4,5</em>-trisphosphate (<em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>) either as pure sonicated lipids or in detergent mixed micelles. When presented as sonicated lipids, <em>PtdIns</em>-4,5-P2 and <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> were equipotent in activating PRK1, and, furthermore, sonicated phosphatidylinositol (<em>PtdIns</em>) and phosphatidylserine (PtdSer) were equally effective. In detergent mixed micelles, <em>PtdIns</em>-4,5-P2 and <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> also showed a similar potency, but <em>PtdIns</em> and PtdSer were 10-fold less effective in this assay. Similarly, PKC-beta 1, -epsilon, and -nu were all activated by <em>PtdIns</em>-4,5-P2 and <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> in detergent mixed micelles. The activation constants for <em>PtdIns</em>-4,5-P2 and <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> were essentially the same for all the kinases tested, implying no specificity in this in vitro analysis. Consistent with this conclusion, the effects of <em>PtdIns</em>-4,5-P2 and <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> were found to be inhibited at 10 mM Mg2+ and mimicked by high concentrations of inositol hexaphosphate and inositol hexasulfate. The similar responses of these two classes of lipid-activated protein kinase to these phosphoinositides are discussed in light of their potential roles as second messengers.

Inositol phosphates are widely produced throughout animal and plant tissues. Diphosphoinositol pentakisphosphate (InsP7) contains an energetic pyrophosphate bond. Here we demonstrate that disruption of inositol hexakisphosphate kinase 1 (InsP6K1), one of the three mammalian inositol hexakisphosphate kinases (InsP6Ks) that convert inositol hexakisphosphate (InsP6) to InsP7, conferred enhanced phosphatidylinositol-(<em>3,4,5</em>)-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>)-mediated membrane translocation of the pleckstrin homology domain of the kinase Akt and thus augmented downstream <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> signaling in mouse neutrophils. Consequently, these neutrophils had greater phagocytic and bactericidal ability and amplified NADPH oxidase-mediated production of superoxide. These phenotypes were replicated in human primary neutrophils with pharmacologically inhibited InsP6Ks. In contrast, an increase in intracellular InsP7 blocked chemoattractant-elicited translocation of the pleckstrin homology domain to the membrane and substantially suppressed <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-mediated cellular events in neutrophils. Our findings establish a role for InsP7 in signal transduction and provide a mechanism for modulating <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> signaling in neutrophils.

The spindle orientation is regulated by the interaction of astral microtubules with the cell cortex. We have previously shown that spindles in nonpolarized adherent cells are oriented parallel to the substratum by an actin cytoskeleton- and phosphatidylinositol <em>3,4,5</em>-triphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>]-dependent mechanism. Here, we show that Cdc42, a Rho family of small GTPases, has an essential role in this mechanism of spindle orientation by regulating both the actin cytoskeleton and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. Knockdown of Cdc42 suppresses PI(3)K activity in M phase and induces spindle misorientation. Moreover, knockdown of Cdc42 disrupts the cortical actin structures in metaphase cells. Our results show that p21-activated kinase 2 (PAK2), a target of Cdc42 and/or Rac1, plays a key role in regulating actin reorganization and spindle orientation downstream from Cdc42. Surprisingly, PAK2 regulates spindle orientation in a kinase activity-independent manner. BetaPix, a guanine nucleotide exchange factor for Rac1 and Cdc42, is shown to mediate this kinase-independent function of PAK2. This study thus demonstrates that spindle orientation in adherent cells is regulated by two distinct pathways downstream from Cdc42 and uncovers a novel role of the Cdc42-PAK2-betaPix-actin pathway for this mechanism.