Inositol polyphosphate multikinase is a physiologic PI3-kinase that activates Akt/PKB

The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP(3) as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP(3) generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.     

Activation of the Akt-related cytokine-independent survival kinase requires interaction of its phox domain with endosomal phosphatidylinositol 3-phosphate

Protein kinases of the Akt and related serum- and glucocorticoid-regulated kinase (SGK) families are major downstream mediators of phosphatidylinositol (PI) 3-kinase signaling to many cellular processes including metabolic flux, membrane trafficking, and apoptosis. Activation of these kinases is thought to occur at the plasma membrane through their serine and threonine phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) protein kinase, which interacts with membrane 3'-polyphosphoinositides through its pleckstrin homology (PH) domain. Here, we demonstrate that the SGK family member cytokine-independent survival kinase (CISK) binds strongly and selectively to the monophosphoinositide PI(3)P through its phox homology (PX) domain. Comparing native green fluorescent protein-CISK (EGFP-CISK) to a mutant EGFP-CISK (Y51A) that displays attenuated binding to PI(3)P reveals that this interaction is both necessary and sufficient for its localization to early endosome antigen (EEA1)-positive endosomes. Furthermore, early endosome association of expressed epitope-tagged CISK in COS cells directed by binding of its PX domain to PI(3)P is required for activation of the CISK protein kinase by both insulin-like growth factor-1 and epidermal growth factor. Taken together, these results reveal a critical role of endosomal PI(3)P in the signal transmission mechanism whereby this survival kinase is activated in response to PI3-kinase stimulation by growth factors.     

3' Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP)

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.     

Phosphorylation-independent dual-site binding of the FHA domain of KIF13 mediates phosphoinositide transport via centaurin alpha1

Phosphatidylinositol 3,4,5-triphosphate (PIP3) plays a key role in neuronal polarization and axon formation. PIP3-containing vesicles are transported to axon tips by the kinesin KIF13B via an adaptor protein, centaurin α1 (CENTA1). KIF13B interacts with CENTA1 through its forkhead-associated (FHA) domain. We solved the crystal structures of CENTA1 in ligand-free, KIF13B-FHA domain-bound, and PIP3 head group (IP4)-bound conformations, and the CENTA1/KIF13B-FHA/IP4 ternary complex. The first pleckstrin homology (PH) domain of CENTA1 specifically binds to PIP3, while the second binds to both PIP3 and phosphatidylinositol 3,4-biphosphate (PI(3,4)P(2)). The FHA domain of KIF13B interacts with the PH1 domain of one CENTA1 molecule and the ArfGAP domain of a second CENTA1 molecule in a threonine phosphorylation-independent fashion. We propose that full-length KIF13B and CENTA1 form heterotetramers that can bind four phosphoinositide molecules in the vesicle and transport it along the microtubule.     

A positive role of phosphatidylinositol 3-kinase in aging phenotype expression in cultured human diploid fibroblasts

In order to detect the role that phosphatidylinositol 3-kinase (PI3K) plays in the aging of human diploid fibroblasts, we analyzed cellular inositol phospholipids and expression of PI3Ks. In aged cells a decrease in phosphatidylinositol 3,4-bisphosphate (PI3,4P(2)) was notable, while phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) decreased slightly. On the other hand, the messages of PI3K IIalpha, Vps34, and p110delta decreased and that of PI3K IIbeta increased during aging. These changes might relate to the aging phenomena, with the PI3K subspecies functioning differentially. Consistently, a PI3K inhibitor LY294002 greatly suppressed enlargement and flattening of cell body and nucleus as well as cell proliferation, both phenotypes being typical of aged cells. An oxidative stress, pulse exposure to hydrogen peroxide (H(2)O(2)), induced these senescent cell-like phenotypes, which LY294002 was also able to abolish. Upon examining three different cell systems (HL-60, N1E-115, and PC-12 cells) we found clear parallelism in a cellular event between the dependence on a PI3K activity and the sensitivity to H(2)O(2). On the analogy of these relationships, we could hypothesize that expression of an aging phenotype such as the morphogenesis is positively promoted by some PI3K subspecies, if such a phenotype as cell cycling is negatively affected by attenuation of another PI3K function in the course of cellular aging.     

PI3K/Akt signaling requires spatial compartmentalization in plasma membrane microdomains

Spatial compartmentalization of signaling pathway components generally defines the specificity and enhances the efficiency of signal transduction. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is known to be compartmentalized within plasma membrane microdomains; however, the underlying mechanisms and functional impact of this compartmentalization are not well understood. Here, we show that phosphoinositide-dependent kinase 1 is activated in membrane rafts in response to growth factors, whereas the negative regulator of the pathway, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is primarily localized in nonraft regions. Alteration of this compartmentalization, either by genetic targeting or ceramide-induced recruitment of PTEN to rafts, abolishes the activity of the entire pathway. These findings reveal critical steps in raft-mediated PI3K/Akt activation and demonstrate the essential role of membrane microdomain compartmentalization in enabling PI3K/Akt signaling. They further suggest that dysregulation of this compartmentalization may underlie pathological complications such as insulin resistance.     

Respiratory burst oxidase activation can be dissociated from phosphatidylinositol bisphosphate degradation in a cell-free system from human neutrophils

Activation of the respiratory burst oxidase in cell-free preparations from 32P-labeled neutrophils was compared with changes in levels of radioactively labeled phosphoinositides in the same preparations. With membrane particles, treatment with sodium dodecyl sulfate (SDS) in the presence of cytosol led to activation of the oxidase without an alteration in levels of labeled phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol 4-phosphate (PIP). Conversely, solubilization of the membrane particles with deoxycholate resulted in loss of nearly 98% of the radioactive PIP2 without activation of the oxidase. In this solubilized preparation, the oxidase could subsequently be fully activated by SDS in the presence of cytosol, even though the labeled PIP2 was almost totally depleted. Two PIP2-derived second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, as well as the protein kinase C activator phorbol myristate acetate (PMA), failed to activate the oxidase. These results suggest that in a cell- free preparation from human neutrophils, detergent-mediated activation of the respiratory burst oxidase is independent of changes in the levels of phosphoinositides or phosphoinositide-derived second messengers.     

Small molecule inhibition of phosphatidylinositol-3,4,5-triphosphate (PIP3) binding to pleckstrin homology domains

The PI3-kinase (PI3K) pathway regulates many cellular processes, especially cell metabolism, cell survival, and apoptosis. Phosphatidylinositol-3,4,5-trisphosphate (PIP3), the product of PI3K activity and a key signaling molecule, acts by recruiting pleckstrin-homology (PH) domain-containing proteins to cell membranes. Here, we describe a new structural class of nonphosphoinositide small molecule antagonists (PITenins, PITs) of PIP3-PH domain interactions (IC(50) ranges from 13.4 to 31 μM in PIP3/Akt PH domain binding assay). PITs inhibit interactions of a number of PIP3-binding PH domains, including those of Akt and PDK1, without affecting several PIP2-selective PH domains. As a result, PITs suppress the PI3K-PDK1-Akt pathway and trigger metabolic stress and apoptosis. A PIT-1 analog displayed significant antitumor activity in vivo, including inhibition of tumor growth and induction of apoptosis. Overall, our studies demonstrate the feasibility of developing specific small molecule antagonists of PIP3 signaling.     

Effect of growth hormone-releasing factor on phosphoinositide hydrolysis in somatotrophs

We studied the role of the phosphatidylinositol system in the action of growth hormone-releasing factor (GRF). We asked whether GRF stimulates the activity of phospholipase C by determining GRF-induced changes in 32P labeling of the individual phosphoinositides and inositol phosphates in purified rat somatotrophs. The somatotrophs were challenged with GRF (10(-7)M) for 0.33, 1, 3, 10, 30, and 90 min. GRF did not significantly or consistently alter 32P incorporation into phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol monophosphate (PIP), or phosphatidylinositol (PI), except for a small reduction in PIP labeling at 90 min. In general the level of 32P incorporation into the inositol phosphates did not increase but instead decreased with GRF. There was a small but significant reduction of labeling of inositol trisphosphate (IP3) at 90 min of GRF incubation. There were also small but significant decreases in 32P incorporation into inositol bisphosphate (IP2) at 0.33, 3, and 30 min. GRF did not significantly alter 32P labeling of inositol monophosphate (IP). These results indicate that GRF does not stimulate phospholipase C activity in somatotrophs. We conclude that the phosphatidylinositol second messenger system does not play an essential role in the action of GRF.     

Deactivation of Akt by a small molecule inhibitor targeting pleckstrin homology domain and facilitating Akt ubiquitination

The phosphatidylinositol-3,4,5-triphosphate (PIP3) binding function of pleckstrin homology (PH) domain is essential for the activation of oncogenic Akt/PKB kinase. Following the PIP3-mediated activation at the membrane, the activated Akt is subjected to other regulatory events, including ubiquitination-mediated deactivation. Here, by identifying and characterizing an allosteric inhibitor, SC66, we show that the facilitated ubiquitination effectively terminates Akt signaling. Mechanistically, SC66 manifests a dual inhibitory activity that directly interferes with the PH domain binding to PIP3 and facilitates Akt ubiquitination. A known PH domain-dependent allosteric inhibitor, which stabilizes Akt, prevents the SC66-induced Akt ubiquitination. A cancer-relevant Akt1 (e17k) mutant is unstable, making it intrinsically sensitive to functional inhibition by SC66 in cellular contexts in which the PI3K inhibition has little inhibitory effect. As a result of its dual inhibitory activity, SC66 manifests a more effective growth suppression of transformed cells that contain a high level of Akt signaling, compared with other inhibitors of PIP3/Akt pathway. Finally, we show the anticancer activity of SC66 by using a soft agar assay as well as a mouse xenograft tumor model. In conclusion, in this study, we not only identify a dual-function Akt inhibitor, but also demonstrate that Akt ubiquitination could be chemically exploited to effectively facilitate its deactivation, thus identifying an avenue for pharmacological intervention in Akt signaling.     

Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors

Stimulation of cells with various peptide growth factors induces the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3) through activation of phosphatidylinositol 3-kinase. The action of this enzyme is reversed by that of the tumor suppressor PTEN. With the use of cells overexpressing NADPH oxidase 1 or peroxiredoxin II, we have now shown that H2O2 produced in response to stimulation of cells with epidermal growth factor or platelet-derived growth factor potentiates PIP3 generation and activation of the protein kinase Akt induced by these growth factors. We also show that a small fraction of PTEN molecules is transiently inactivated as a result of oxidation of the essential cysteine residue of this phosphatase in various cell types stimulated with epidermal growth factor, platelet-derived growth factor, or insulin. These results suggest that the activation of phosphatidylinositol 3-kinase by growth factors might not be sufficient to induce the accumulation of PIP3 because of the opposing activity of PTEN and that the concomitant local inactivation of PTEN by H2O2 might be needed to increase the concentration of PIP3 sufficiently to trigger downstream signaling events. Furthermore, together with previous observations, our data indicate that peroxiredoxin likely participates in PIP3 signaling by modulating the local concentration of H2O2.     

Negative feedback regulation of Rac in leukocytes from mice expressing a constitutively active phosphatidylinositol 3-kinase gamma

Polarization of chemotaxing cells depends on positive feedback loops that amplify shallow gradients of chemoattractants into sharp intracellular responses. In particular, reciprocal activation of phosphatidylinositol 3-kinases (PI3Ks) and small GTPases like Rac leads to accumulation, at the leading edge, of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3). Mice carrying a "knockin" allele of the G protein-coupled receptor (GPCR)-activated PI3Kgamma, encoding a plasma membrane-targeted protein appeared normal, but their leukocytes showed GPCR-uncoupled PIP3 accumulation. In vivo, the mutation increased proliferation and decreased apoptosis, leading to leukocytosis and delayed resolution of inflammation in wound healing. Mutant leukocytes showed significantly impaired directional cell migration in response to chemoattractants. Stimulated mutant macrophages did not polarize PIP3 and showed a shortened Rac activation because of enhanced PI3K-dependent activation of RacGAPs. Together with the finding that chemoattractants stimulate a PIP3-dependent GAP activation in wild-type macrophages, these results identify a molecular mechanism involving PI3K- and RacGAP-dependent negative control of Rac that limits and fine-tunes feedback loops promoting cell polarization and directional motility.     

Diffusion-limited phase separation in eukaryotic chemotaxis

The ability of cells to sense spatial gradients of chemoattractant factors governs the development of complex eukaryotic organisms. Cells exposed to shallow chemoattractant gradients respond with strong accumulation of the enzyme phosphatidylinositol 3-kinase (PI3K) and its D3-phosphoinositide product (PIP(3)) on the plasma membrane side exposed to the highest chemoattractant concentration, whereas PIP(3)-degrading enzyme PTEN and its product PIP(2) localize in a complementary pattern. Such an early symmetry-breaking event is a mandatory step for directed cell movement elicited by chemoattractants, but its physical origin is still mysterious. Here, we propose that directional sensing is the consequence of a phase-ordering process mediated by phosphoinositide diffusion and driven by the distribution of chemotactic signal. By studying a realistic reaction-diffusion lattice model that describes PI3K and PTEN enzymatic activity, recruitment to the plasma membrane, and diffusion of their phosphoinositide products, we show that the effective enzyme-enzyme interaction induced by catalysis and diffusion introduces an instability of the system toward phase separation for realistic values of physical parameters. In this framework, large reversible amplification of shallow chemotactic gradients, selective localization of chemical factors, macroscopic response timescales, and spontaneous polarization arise naturally. The model is robust with respect to order-of-magnitude variations of the parameters.     

FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization

Zea mays plasma membrane intrinsic proteins (ZmPIPs) fall into two groups, ZmPIP1s and ZmPIP2s, that exhibit different water channel activities when expressed in Xenopus oocytes. ZmPIP1s are inactive, whereas ZmPIP2s induce a marked increase in the membrane osmotic water permeability coefficient, P(f). We previously showed that, in Xenopus oocytes, ZmPIP1;2 and ZmPIP2;1 interact to increase the cell P(f). Here, we report the localization and interaction of ZmPIP1s and ZmPIP2s in living maize cells. ZmPIPs were fused to monomeric yellow fluorescent protein and/or monomeric cyan fluorescent protein and expressed transiently in maize mesophyll protoplasts. When expressed alone, ZmPIP1 fusion proteins were retained in the endoplasmic reticulum, whereas ZmPIP2s were found in the plasma membrane. Interestingly, when coexpressed with ZmPIP2s, ZmPIP1s were relocalized to the plasma membrane. Using FRET/fluorescence lifetime imaging microscopy, we demonstrated that this relocalization results from interaction between ZmPIP1s and ZmPIP2s. Immunoprecipitation experiments provided additional evidence for the association of ZmPIP1;2 and ZmPIP2;1 in maize roots and suspension cells. These data suggest that PIP1-PIP2 interaction is required for in planta PIP1 trafficking to the plasma membrane to modulate plasma membrane permeability.     

SHIP inhibits Akt activation in B cells through regulation of Akt membrane localization

Activation of the serine/threonine kinase Akt and the regulation of its activation are recognized as critical in controlling proliferative/survival signals via many hematopoietic receptors. In B lymphocytes, the B-cell receptor (BCR)-mediated activation of Akt is attenuated by co-cross-linking of BCR with the inhibitory receptor Fc gamma RIIB1, and the binding of the SH2 domain-containing inositol phosphatase, SHIP, to Fc gamma RIIB1. Because SHIP dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PIP3) and activation of Akt requires PIP3, the destruction of this phospholipid has been proposed as the mechanism for Akt inhibition. However, upstream kinases that activate Akt, such as PDK1, also require PIP3 for activation. In this report, we addressed whether SHIP inhibits Akt directly at the level of Akt recruitment to the membrane, indirectly through PDK recruitment/phosphorylation of Akt, or both. We generated stable B-cell lines expressing a regulatable, but constitutively membrane-bound Akt that still required PDK-dependent phosphorylation for activation. Several lines of evidence suggested that activation of this membrane-targeted Akt is not inhibited by Fc gamma RIIB1/SHIP and that PDK is not a target for SHIP-mediated inhibition. These data demonstrate that SHIP inhibits Akt primarily through regulation of Akt membrane localization. We also observed during these studies that Fc gamma RIIB1/SHIP does not inhibit p70(S6k) activation, even though several other PIP3-dependent events were down-regulated. Because the enhanced activation of Akt in the absence of SHIP correlates with hyperproliferation in the myeloid lineage, our data have implications for SHIP and Akt-dependent regulation of proliferation in the hematopoietic lineage. (Blood. 2000;96:1449-1456)     

The inositol polyphosphate 4-phosphatase forms a complex with phosphatidylinositol 3-kinase in human platelet cytosol

Inositol polyphosphate 4-phosphatase (4-phosphatase) is an enzyme that catalyses the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. In human platelets the formation of this phosphatidylinositol, by the actions of phosphatidylinositol 3-kinase (PI 3-kinase), correlates with irreversible platelet aggregation. We have shown previously that a phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase forms a complex with the p85 subunit of PI 3-kinase. In this study we investigated whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contained 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contained PI 3-kinase enzyme activity and an 85-kDa polypeptide recognized by antibodies to the p85 adapter subunit of PI 3-kinase. After thrombin activation, the 4-phosphatase translocated to the actin cytoskeleton along with PI 3-kinase in an integrin- and aggregation-dependent manner. The majority of the PI 3-kinase/4-phosphatase complex (75%) remained in the cytosolic fraction. We propose that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4)P2 production.     

Arf6: a new player in FcgammaRIIIA lymphocyte-mediated cytotoxicity

The activation of phosphoinositide metabolism represents a critical step in the signaling pathways leading to the activation of cytolytic machinery, but its regulation is partially understood. We report here that the stimulation of the low-affinity receptor for immunoglobulin G (IgG) (FcgammaRIIIA, CD16) on primary human natural killer (NK) cells induces a phosphatidylinositol 3-kinase (PI3K)-dependent activation of the small G protein Arf6. We first demonstrate a functional role for Arf6-dependent signals in the activation of the antibody-dependent cellular cytotoxicity (ADCC) attributable to the control of secretion of lytic granule content. We also show that Arf6 couples CD16 to the lipid-modifying enzymes phosphatidylinositol4phosphate 5-kinase type I alpha (PI5KIalpha) and phospholipase D (PLD) that are involved in the control of granule secretion; Arf6, but not Rho family small G proteins RhoA and Rac1, is required for receptor-induced PI5KIalpha membrane targeting as well as for PI5KIalpha and PLD activation. Our findings suggest that Arf6 plays a crucial role in the generation of a phosphatidylinositol4,5-bisphosphate (PIP2) plasma membrane pool required for cytolytic granule-mediated target cell killing.     

Putative role of the phosphatidylinositol 3-kinase-Akt signaling pathway in the survival of granulosa cells

Insulin-like growth factor-1 (IGF-1) is an important differentiation and survival factor for granulosa cells. The purpose of this study was to test the hypothesis that IGF-1 promotes survival of porcine granulosa cells by signaling though the phosphatidylinositol (PI) 3-kinase/Akt signal transduction pathway. Treatment with IGF-1 (100 ng/ml) for 10 min stimulated PI 3-kinase and Akt protein kinase activity. IGF-I stimulated the phosphorylation and activation of Akt in a time- and concentration-dependent manner. The PI 3-kinase inhibitors wortmannin and LY294002 blocked IGF-1 induced increases in PI 3-kinase activity and phosphorylation of Akt. Additionally, IGF-1 treatment prevented apoptosis. The survival response to IGF-I was blocked by treatment with either wortmannin or LY294002. These data suggest that IGF-I-induced phosphorylation of Akt is mediated through PI 3-kinase and that inactivation of this pathway results in granulosa cell apoptosis. We conclude that the P1 3-kinase/Akt signaling serves as a functional survival pathway in the ovary.     

Akt2, phosphatidylinositol 3-kinase, and PTEN are in lipid rafts of intestinal cells: role in absorption and differentiation

BACKGROUND AND AIMS: In intestinal Na absorptive cells, phosphatidylinositol 3-kinase (PI 3-K) is involved in rapid epidermal growth factor (EGF) stimulation of Na absorption by the brush border membrane (BBM) Na(+)/H(+) exchanger NHE3. However, how NHE3 is regulated by the PI 3-K pathway and the role of Akt2 are poorly defined. METHODS: The localization of Akt, PI 3-K, and NHE3 was determined by either immunocytochemistry and/or membrane fractionation using OptiPrep density gradient centrifugation. RESULTS: In ileum, active total Akt was present most in the villi and basal layer of the crypts, and Akt2 was mostly in villi. In villus cells, PI 3-K and Akt2 were mostly at the apical surface at which they were present partially in lipid rafts (LR). EGF increased PI 3-K and active Akt2 in ileal BBM at the same time that it increased PI 3-K-dependent trafficking of NHE3 to BBM and stimulation of Na absorption. However, Akt2 was only active in the detergent soluble (DS) pool and not LR of ileal BBM, which correlated with the presence of PTEN in LR. In Caco-2 cells, while EGF stimulated BB NHE3, Akt2 was active in both LR and DS pools. This correlated with the lack of PTEN in the LR of Caco-2 membranes. Akt2 also correlated with epithelial cell differentiation. Akt2 amount and activity were greater in differentiated than undifferentiated Caco-2 cells. CONCLUSIONS: These results suggest that LR may play an important role in determining the function of PI 3-K/Akt2 signaling, including stimulation of intestinal Na absorption. These results also suggest that LR-associated Akt2 may be involved in enterocyte differentiation.     

Neutrophil spontaneous death is mediated by down-regulation of autocrine signaling through GPCR,PI3Kγ, ROS,and actin

Neutrophil spontaneous apoptosis plays a crucial role in neutrophil homeostasis and the resolution of inflammation. We previously established Akt deactivation as a key mediator of this tightly regulated cellular death program. Nevertheless, the molecular mechanisms governing the diminished Akt activation were not characterized. Here, we report that Akt deactivation during the course of neutrophil spontaneous death was a result of reduced PtdIns(3,4,5)P3 level. The phosphatidylinositol lipid kinase activity of PI3Kγ, but not class IA PI3Ks, was significantly reduced during neutrophil death. The production of PtdIns(3,4,5)P3 in apoptotic neutrophils was mainly maintained by autocrinely released chemokines that elicited PI3Kγ activation via G protein–coupled receptors. Unlike in other cell types, serum-derived growth factors did not provide any survival advantage in neutrophils. PI3Kγ, but not class IA PI3Ks, was negatively regulated by gradually accumulated ROS in apoptotic neutrophils, which suppressed PI3Kγ activity by inhibiting an actin-mediated positive feedback loop. Taken together, these results provide insight into the mechanism of neutrophil spontaneous death and reveal a cellular pathway that regulates PtdIns(3,4,5)P3/Akt in neutrophils.