PI3K signaling controls cell fate at many points in B lymphocyte development and activation

Many receptors on diverse cell types activate phosphoinositide 3-kinase (PI3K). The lipid products of PI3K, termed 3-phosphoinositides, regulate numerous cellular processes by recruiting specific proteins to membrane signaling complexes. In the B lymphocyte lineage, PI3K activation is a critical control point at various stages of development, proliferation and differentiation. PI3K signaling is promoted by stimulatory receptors such as surface immunoglobulin, CD40, Toll-like receptors and cytokine receptors, and opposed by the inhibitory receptor FcgammaRIIB1. Genetic dissection of the PI3K pathway in mice has indicated that certain B cell functions are regulated by a limited set of PI3K isoforms and downstream effectors. Here we review our current understanding of how signals are relayed to and from PI3K in B cells.     

Platelet-derived growth factor-dependent activation of phosphatidylinositol 3-kinase is regulated by receptor binding of SH2-domain-containing proteins which influence Ras activity.

Upon binding of platelet-derived growth factor (PDGF), the PDGF beta receptor (PDGFR) undergoes autophosphorylation on distinct tyrosine residues and binds several SH2-domain-containing signal relay enzymes, including phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein of Ras (RasGAP), and the tyrosine phosphatase SHP-2. In this study, we have investigated whether PDGF-dependent PI3K activation is affected by the other proteins that associate with the PDGFR. We constructed and characterized a series of PDGFR mutants which contain binding sites for PI3K as well as one additional protein, either RasGAP, SHP-2, or PLC gamma. While all of the receptors had wild-type levels of PDGF-stimulated tyrosine kinase activity and associated with comparable amounts of PI3K activity, their abilities to trigger accumulation of PI3K products in vivo differed dramatically. The wild-type receptor, as well as receptors that recruited PI3K or PI3K and SHP-2, were all capable of fully activating PI3K. In contrast, receptors that associated with PI3K and RasGAP or PI3K and PLC gamma displayed a greatly reduced ability to stimulate production of PI3K products. When this series of receptors was tested for their ability to activate Ras, we observed a strong positive correlation between Ras activation and PI3K activation. Further investigation of the relationship between Ras and PI3K indicated that Ras was upstream of PI3K. Thus, activation of PI3K requires not only binding of PI3K to the tyrosine-phosphorylated PDGFR but accumulation of GTP-bound Ras as well. Furthermore, PLC gamma and RasGAP negatively modulate PDGF-dependent PI3K activation. Finally, PDGF-stimulated signal relay can be regulated by altering the ratio of SH2-domain-containing enzymes that are recruited to the PDGFR.     

Phosphatidylinositol 3-Kinase Is a Negative Regulator of Cellular Differentiation

Phosphatidylinositol 3-kinase (PI3K) has been shown to be an important mediator of intracellular signal transduction in mammalian cells. We show here, for the first time, that the blockade of PI3K activity in human fetal undifferentiated cells induced morphological and functional endocrine differentiation. This was associated with an increase in mRNA levels of insulin, glucagon, and somatostatin, as well as an increase in the insulin protein content and secretion in response to secretagogues. Blockade of PI3K also increased the proportion of pluripotent precursor cells coexpressing multiple hormones and the total number of terminally differentiated cells originating from these precursor cells. We examined whether any of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in fetal islet cells. The activity of PI3K was inversely correlated with the hepatocyte growth factor/scatter factor–induced downregulation or nicotinamideinduced upregulation of islet-specific gene expression, giving support to the role of PI3K, as a negative regulator of endocrine differentiation. In conclusion, our results provide a mechanism for the regulation of hormone-specific gene expression during human fetal neogenesis. They also suggest a novel function for PI3K, as a negative regulator of cellular differentiation.     

Solubilization and characterization of active somatostatin receptors from rat pancreas

Somatostatin receptors were solubilized from rat pancreatic membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS). The binding of an iodinated somatostatin analog [125I-Tyr3]SMS to the soluble fraction was time-dependent, saturable, and reversible. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of somatostatin binding sites with a Kd of 0.3 nM and a Bmax of 210 fmol/mg. As observed with membrane-bound receptors, soluble binding receptors were sensitive to the GTP analog GTP gamma S indicating that they are functionally linked to a G protein. A molecular weight of about 400,000 was determined for soluble receptors under native conditions by gel filtration. In denaturing gel electrophoresis, photoaffinity labeling of soluble receptors identified a major protein of Mr = 100,000 and two minor proteins of Mr = 56,000 and 21,000. Isoelectric focusing of soluble receptors revealed that the somatostatin receptor is an acidic protein with pI 4.8. The soluble somatostatin receptor is a glycoprotein which can be specifically bound to the wheat germ agglutinin lectin and eluted by triacetyl-chitotriose.     

G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility

Phosphoinositide 3-kinase (PI3K)gamma and Dictyostelium PI3K are activated via G protein-coupled receptors through binding to the Gbetagamma subunit and Ras. However, the mechanistic role(s) of Gbetagamma and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gbeta null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P(3). Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras-guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton.     

A switch of G protein-coupled receptor binding preference from phosphoinositide 3-kinase (PI3K)-p85 to filamin A negatively controls the PI3K pathway

Frequent oncogenic alterations occur in the phosphoinositide 3-kinase (PI3K) pathway, urging identification of novel negative controls. We previously reported an original mechanism for restraining PI3K activity, controlled by the somatostatin G protein-coupled receptor (GPCR) sst2 and involving a ligand-regulated interaction between sst2 with the PI3K regulatory p85 subunit. We here identify the scaffolding protein filamin A (FLNA) as a critical player regulating the dynamic of this complex. A preexisting sst2-p85 complex, which was shown to account for a significant basal PI3K activity in the absence of ligand, is disrupted upon sst2 activation. FLNA was here identified as a competitor of p85 for direct binding to two juxtaposed sites on sst2. Switching of GPCR binding preference from p85 toward FLNA is determined by changes in the tyrosine phosphorylation of p85- and FLNA-binding sites on sst2 upon activation. It results in the disruption of the sst2-p85 complex and the subsequent inhibition of PI3K. Knocking down FLNA expression, or abrogating FLNA recruitment to sst2, reversed the inhibition of PI3K and of tumor growth induced by sst2. Importantly, we report that this FLNA inhibitory control on PI3K can be generalized to another GPCR, the mu opioid receptor, thereby providing an unprecedented mechanism underlying GPCR-negative control on PI3K.     

PI3K Contributed to Modulation of Spinal Nociceptive Information Related to ephrinBs/EphBs

There is accumulating evidence to implicate the importance of EphBs receptors and ephrinBs ligands were involved in modulation of spinal nociceptive information. However, the downstream mechanisms that control this process are not well understood. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K), as the downstream effectors, participates in modulation of spinal nociceptive information related to ephrinBs/EphBs. Intrathecal injection of ephrinB1-Fc produced a dose- and time-dependent thermal and mechanical hyperalgesia, accompanied by the increase of spinal PI3K-p110γ, phosphorylation of AKT (p-AKT) and c-Fos expression. Pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented activation of spinal AKT induced by ephrinB1-Fc. Inhibition of spinal PI3K signaling dose-dependently prevented and reversed pain behaviors and spinal c-Fos protein expression induced by intrathecal injection of ephrinB1-Fc. Inhibition of EphBs receptors by intrathecal injection of EphB1-Fc reduced formalin-induced inflammation and chronic constrictive injury-induced neuropathic pain behaviors accompanied by decreased expression of spinal PI3K,p-AKT and c-Fos protein. Furthermore, pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented ephrinB1-Fc-induced ERK activation in spinal. These data demonstrated that PI3K and PI3K crosstalk to ERK signaling contributed to modulation of spinal nociceptive information related to ephrinBs/EphBs.     

Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival

The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.     

Role of phosphatidylinositol 3-kinase in anti-IgM- and anti-IgD-induced apoptosis in B cell lymphomas

Cross-linking of surface Ig receptors with anti IgM (anti-mu heavy chain, anti-mu), but not anti-IgD (anti-delta heavy chain, anti-delta), Abs leads to growth arrest and apoptosis in several extensively characterized B cell lymphomas. By poorly understood mechanisms, both Igs transiently stimulate c-Myc protein expression. However, ultimately, only anti-mu causes a severe loss in c-Myc and a large induction of p27(Kip1) protein expression. Because phosphatidylinositol 3-kinase (PI3K) has been established as a major modulator of cellular growth and survival, we investigated its role in mediating anti-Ig-stimulated outcomes. Herein, we show that PI3K pathways regulate cell cycle progression and apoptosis in the ECH408 B cell lymphoma. Anti-mu and anti-delta driven c-Myc protein changes precisely follow their effects on the PI3K effector, p70(S6K). Upstream of p70(S6K), signaling through both Ig receptors depresses PI3K pathway phospholipids below control with time, which is followed by p27(Kip1) induction. Conversely, anti-delta, but not anti-mu stimulated PI3K-dependent phospholipid return to control levels by 4-8 h. Abrogation of the PI3K pathway with specific inhibitors mimics anti-mu action, potentiates anti-mu-induced cell death and, importantly, converts anti-delta to a death signal. Transfection with active PI3K kinase construct induces anti-mu resistance, whereas transfection with dominant negative PI3K augments anti-mu sensitivity. Our results show that prolonged disengagement of PI3K or down-regulation of its products by anti-mu (and not anti-delta) determines B cell fate.     

Regulation and roles of PI3Kβ, a major actor in platelet signaling and functions

Phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes involved in the regulation of a number of critical cell functions. Significant progress has been made during the last few years in defining the implication of individual PI3K isoforms. The role of the class IA PI3Kβ in different cell types has only been recently uncovered by the use of isoform-selective inhibitors and the development of mouse models harboring p110β catalytic subunit knock-out or germline knock-in of a kinase-dead allele of p110β. Although it is classically admitted that class IA PI3Ks are activated by receptor tyrosine kinases through recruitment of the regulatory subunits to specific tyrosine phosphorylated motifs via their SH2 domains, PI3Kβ is activated downstream of G protein-coupled receptors, and by co-operation between heterotrimeric G proteins and tyrosine kinases. PI3Kβ has been extensively studied in platelets where it appears to play an important role downstream of ITAM signaling, G protein-coupled receptors and aIIbβ3 integrin. Accordingly, mouse exhibiting p110β inactivation selectively in megakaryocyte/platelets are resistant to thromboembolism induced by carotid injury. The present review summarizes recent data concerning the mechanisms of PI3Kβ regulation and the roles of this PI3K isoform in blood platelet functions and other cell types.     

Activation of phosphoinositide 3-kinase/PKB pathway by CB(1) and CB(2) cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction

Cannabinoids exert a variety of physiological and pharmacological responses in humans through interaction with specific cannabinoid receptors. Cannabinoid receptors described to date belong to the seven-transmembrane-domain receptor superfamily and are coupled through the inhibitory G(i) protein to adenylyl cyclase inhibition. However, downstream signal transduction mechanisms triggered by cannabinoids are poorly understood. We examined here the involvement of the phosphoinositide 3-kinase (PI3K)/PKB pathway in the mechanism of action of cannabinoids in human prostate epithelial PC-3 cells. Cannabinoid receptors CB(1) and CB(2) are expressed in these cells, as shown by RT-PCR, Western blot and immunofluorescence techniques. Treatment of PC-3 cells with either Delta(9)-tetrahydrocannabinol (THC), the major psychoactive ingredient of marijuana, or R-(+)-methanandamide (MET), an analogue of the endogenous cannabinoid anandamide, increased phosphorylation of PKB in Thr308 and Ser473. The stimulation of PKB induced by cannabinoids was blocked by the two cannabinoid receptor antagonists, SR 141716 and SR 144528, and by the PI3K inhibitor LY 294002. These results indicate that activation of cannabinoid receptors in PC-3 cells stimulate the PI3K/PKB pathway. We further investigated the involvement of Raf-1/Erk activation in the mechanism of action of cannabinoid receptors. THC and MET induced translocation of Raf-1 to the membrane and phosphorylation of p44/42 Erk kinase, which was reversed by cannabinoid receptor antagonists and PI3K inhibitor. These results point to a sequential connection between cannabinoid receptors/PI3K/PKB pathway and Raf-1/Erk in prostate PC-3 cells. We also show that this pathway is involved in the mechanism of NGF induction exerted by cannabinoids in PC-3 cells.     

Activated G alpha q inhibits p110 alpha phosphatidylinositol 3-kinase and Akt

Some Gq-coupled receptors have been shown to antagonize growth factor activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. We used a constitutively active Galphaq(Q209L) mutant to explore the effects of Galphaq activation on signaling through the PI3K/Akt pathway. Transient expression of Galphaq(Q209L) in Rat-1 fibroblasts inhibited Akt activation induced by platelet-derived growth factor or insulin treatment. Expression of Galphaq(Q209L) also attenuated Akt activation promoted by coexpression of constitutively active PI3K in human embryonic kidney 293 cells. Galphaq(Q209L) had no effect on the activity of an Akt mutant in which the two regulatory phosphorylation sites were changed to acidic amino acids. Inducible expression of Galphaq(Q209L) in a stably transfected 293 cell line caused a decrease in PI3K activity in p110alpha (but not p110beta) immunoprecipitates. Receptor activation of Galphaq also selectively inhibited PI3K activity in p110alpha immunoprecipitates. Active Galphaq still inhibited PI3K/Akt in cells pretreated with the phospholipase C inhibitor U73122. Finally, Galphaq(Q209L) co-immunoprecipitated with the p110alpha-p85alpha PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated Galphaq(Q209L) with purified recombinant p110alpha-p85alpha in vitro led to a decrease in PI3K activity. These results suggest that agonist binding to Gq-coupled receptors blocks Akt activation via the release of active Galphaq subunits that inhibit PI3K. The inhibitory mechanism seems to be independent of phospholipase C activation and might involve an inhibitory interaction between Galphaq and p110alpha PI3K.     

Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs

Many G protein coupled receptors (GPCRs) cause phosphorylation of MAP kinases through transactivation of the epidermal growth factor receptor (EGF-R), leading to increased cell survival and growth, motility, and migration. Phosphoinositide 3-kinase (PI3K) is one of the important cell survival signaling molecules activated by EGF-R stimulation. However, the extent to which EGF-R transactivation is essential for GPCR agonist-stimulated PI3K activation is not known. Here we examined the mechanism of PI3K activation that elicits GPCR-mediated ERK1/2 activation by pathways dependent and/or independent of EGF-R transactivation in specific cell types. Immortalized hypothalamic neurons (GT1-7 cells) express endogenous gonadotropin-releasing hormone receptors (GnRH-R) and their stimulation causes marked phosphorylation of ERK1/2 and Akt (Ser 473) through transactivation of the EGF-R and recruitment of PI3K. In C9 hepatocytes, agonist activation of AT1 angiotensin II (AT1-R), lysophosphatidic acid (LPA), and EGF receptors caused phosphorylation of Akt through activation of the EGF-R in a PI3K-dependent manner. However, ERK1/2 activation by these agonists in these cells was independent of PI3K activation. In contrast, agonist stimulation of HEK 293 cells stably expressing AT1-R caused ERK1/2 phosphorylation that was independent of EGF-R transactivation but required PI3K activation. LPA signaling in these cells showed partial and complete dependence on EGF-R and PI3K, respectively. These data indicate that GPCR-induced ERK1/2 phosphorylation is dependent or independent of PI3K in specific cell types, and that the involvement of PI3K during ERK1/2 activation is not dependent solely on agonist-induced transactivation of the EGF-R.     

Phosphorylation of the PDGF receptor beta subunit creates a tight binding site for phosphatidylinositol 3 kinase.

The beta subunit of the platelet derived growth factor receptor (PDGFR) coprecipitates with a phosphatidyl-inositol 3 kinase activity (PI3K) following stimulation of cells by PDGF. Mutagenesis of a tyrosine (Y) phosphorylation site, Y751, in the PDGFR, greatly reduces PI3K, consistent with the possibility that phosphorylation of Y751 signals association of PI3K. To test this we have reconstituted the binding of the PDGFR beta subunit and PI3K in vitro. Binding is rapid, saturable and requires phosphorylation of the PDGFR at Y751, but does not require PDGF-dependent phosphorylation of PI3K. To test which portions of the PDGFR are important for binding, we used an antibody to a small region of the receptor that includes Y751. This antibody blocked in vitro binding of PI3K to the receptor, while an antiserum to the C-terminus of the receptor had no effect on binding of PI3K. In addition, we found that PDGF stimulation of a cell results in the association of essentially all the PI3K activity with cellular PDGFRs. These data suggest that PI3K is a specific ligand for PDGF receptors that are phosphorylated at Y751.     

Phosphatidylinositol 3-kinase activation is required for stress protocol-induced modification of hippocampal synaptic plasticity

Stress dramatically affects the induction of hippocampal synaptic plasticity; however, the molecular details of how it does so remain unclear. Phosphatidylinositol 3-kinase (PI3K) signaling plays a crucial role in promoting neuronal survival and neuroplasticity, but its role, if any, in stress-induced alterations of long term potentiation (LTP) and long term depression (LTD) is unknown. We found here that inhibitors of PI3K signaling blocked the effects of acute restraint-tail shock stress protocol on LTP and LTD. Therefore, the purpose of the present study is to explore the signaling events involving PI3K in terms of its role in mediating stress protocol-induced alterations of LTP and LTD. We found that stress protocol-induced PI3K activation can be blocked by various inhibitors, including RU38486 for glucocorticoid receptors, LY294002 for PI3K, and dl-2-amino-5-phosphonopentanoic acid for N-methyl-D-aspartate receptors or brain-derived neurotrophic factor antisense oligonucleotides. Also, immunoblotting analyses revealed that stress protocol induced a profound and prolonged phosphorylation of numbers of PI3K downstream effectors, including 3-phosphoinositide-dependent protein kinase-1, protein kinase B, mammalian target of rapamycin (mTOR), p70 S6 kinase, and eukaryotic initiation factor 4B in hippocampal CA1 homogenate, which was prevented by the PI3K inhibitor pretreatment. More importantly, we found that stress protocol significantly increased the protein expression of dendritic scaffolding protein PSD-95 (postsynaptic density-95), which is known to be involved in LTP and LTD, in an mTOR-dependent manner. These results identify a key role of PI3K signaling in mediating the stress protocol-induced modification of hippocampal synaptic plasticity and further suggest that PI3K may do so by invoking the protein expression of PSD-95.     

Activating Mutations of the Serotonin 5-HT2C Receptor

Abstract: Site-directed mutagenesis was performed to create a mutant serotonin 5-HT_2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS-7 cells, the binding affinity of 5-HT for [³H]-mesulergine-labeled 5-HT_2C receptors increased from 203 n M (native) to 76 n M for S312F and 6.6 n M for S312K mutant receptors. 5-HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 n M (native) to 28 n M for S312F and 2.7 n M for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five-fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [³H]5-HT and [³H]-mesulergine labeled the same number of S312K mutant receptors and 5'-guanylylimidodiphosphate had no effect on [³H]5-HT binding. These results indicate that serine → lysine mutation at amino acid no. 312 produces an agonist high-affinity state of the 5-HT_2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.     

Signal transduction at point-blank range: analysis of a spatial coupling mechanism for pathway crosstalk

The plasma membrane provides a physical platform for the orchestration of molecular interactions and biochemical conversions involved in the early stages of receptor-mediated signal transduction in living cells. In that context, we introduce here the concept of spatial coupling, wherein simultaneous recruitment of different enzymes to the same receptor scaffold facilitates crosstalk between different signaling pathways through the local release and capture of activated signaling molecules. To study the spatiotemporal dynamics of this mechanism, we have developed a Brownian dynamics modeling approach and applied it to the receptor-mediated activation of Ras and the cooperative recruitment of phosphoinositide 3-kinase (PI3K) by activated receptors and Ras. Various analyses of the model simulations show that cooperative assembly of multimolecular complexes nucleated by activated receptors is facilitated by the local release and capture of membrane-anchored signaling molecules (such as active Ras) from/by receptor-bound signaling proteins. In the case of Ras/PI3K crosstalk, the model predicts that PI3K is more likely to be recruited by activated receptors bound or recently visited by the enzyme that activates Ras. By this mechanism, receptor-bound PI3K is stabilized through short-range, diffusion-controlled capture of active Ras and Ras/PI3K complexes released from the receptor complex. We contend that this mechanism is a means by which signaling pathways are propagated and spatially coordinated for efficient crosstalk between them.     

A novel role for phosphatidylinositol 3-kinase beta in signaling from G protein-coupled receptors to Akt

The protein kinase Akt plays a central role in a number of key biological functions including protein synthesis, glucose homeostasis, and the regulation of cell survival or death. The mechanism by which tyrosine kinase growth factor receptors stimulate Akt has been recently defined. In contrast, the mechanism of activation of Akt by other cell surface receptors is much less understood. For G protein-coupled receptors (GPCRs), conflicting data suggest that these receptors stimulate Akt in a cell type-specific manner by a yet to be fully elucidated mechanism. Here, we took advantage of the availability of cells, where Akt activity could not be enhanced by agonists acting on this large family of cell surface receptors, such as NIH 3T3 cells, to investigate the pathway linking GPCRs to Akt. We present evidence that expression of phosphatidylinositol 3-kinase (PI3K) beta is necessary and sufficient to transmit signals from G proteins to Akt in these murine fibroblasts and that the activation of PI3Kbeta may represent the most likely mechanism whereby GPCRs stimulate Akt, as the vast majority of cells do not express PI3Kgamma, a known G protein-sensitive PI3K isoform. Furthermore, available evidence indicates that GPCRs activate Akt by a pathway distinct from that utilized by growth factor receptors, as it involves the tyrosine phosphorylation-independent activation of PI3Kbeta by G protein betagamma dimers.     

Direct binding of p85 to sst2 somatostatin receptor reveals a novel mechanism for inhibiting PI3K pathway

Phosphatidylinositol 3-kinase (PI3K) regulates many cellular functions including growth and survival, and its excessive activation is a hallmark of cancer. Somatostatin, acting through its G protein-coupled receptor (GPCR) sst2, has potent proapoptotic and anti-invasive activities on normal and cancer cells. Here, we report a novel mechanism for inhibiting PI3K activity. Somatostatin, acting through sst2, inhibits PI3K activity by disrupting a pre-existing complex comprising the sst2 receptor and the p85 PI3K regulatory subunit. Surface plasmon resonance and molecular modeling identified the phosphorylated-Y71 residue of a p85-binding pYXXM motif in the first sst2 intracellular loop, and p85 COOH-terminal SH2 as direct interacting domains. Somatostatin-mediated dissociation of this complex as well as p85 tyrosine dephosphorylation correlates with sst2 tyrosine dephosphorylation on the Y71 residue. Mutating sst2-Y71 disabled sst2 to interact with p85 and somatostatin to inhibit PI3K, consequently abrogating sst2's ability to suppress cell survival and tumor growth. These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand-activated GPCR of PI3K-dependent survival pathways, which may be an important molecular target for antineoplastic therapy.     

Ras‐dependent and Ras‐independent effects of PI3K in Drosophila motor neurons

The lipid kinase PI3K plays key roles in cellular responses to activation of receptor tyrosine kinases or G protein coupled receptors such as the metabotropic glutamate receptor (mGluR). Activation of the PI3K catalytic subunit p110 occurs when the PI3K regulatory subunit p85 binds to phosphotyrosine residues present in upstream activating proteins. In addition, Ras is uniquely capable of activating PI3K in a p85‐independent manner by binding to p110 at amino acids distinct from those recognized by p85. Because Ras, like p85, is activated by phosphotyrosines in upstream activators, it can be difficult to determine if particular PI3K‐dependent processes require p85 or Ras. Here, we ask if PI3K requires Ras activity for either of two different PI3K‐regulated processes within Drosophila larval motor neurons. To address this question, we determined the effects on each process of transgenes and chromosomal mutations that decrease Ras activity, or mutations that eliminate the ability of PI3K to respond to activated Ras. We found that PI3K requires Ras activity to decrease motor neuron excitability, an effect mediated by ligand activation of the single Drosophila mGluR DmGluRA. In contrast, the ability of PI3K to increase nerve terminal growth is Ras‐independent. These results suggest that distinct regulatory mechanisms underlie the effects of PI3K on distinct phenotypic outputs.