The p85alpha regulatory subunit of class IA phosphoinositide 3-kinase regulates beta-selection in thymocyte development

We examined the role of class IA PI3K in pre-TCR controlled beta-selection and TCR-controlled positive/negative selection in thymic development. Using mice deficient for p85alpha, a major regulatory subunit of the class IA PI3K family, the role of class IA PI3K in beta-selection was examined by injection of anti-CD3epsilon mAb into p85alpha(-/-)Rag-2(-/-) mice, which mimics pre-TCR signals. Transition of CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP) thymocytes triggered by anti-CD3epsilon mAb was significantly impaired in p85alpha(-/-)Rag-2(-/-) compared with p85alpha(+/-)Rag-2(-/-) mice. Furthermore, DP cell numbers were lower in p85alpha(-/-)DO11.10/Rag-2(-/-) TCR-transgenic mice than in DO11.10/Rag-2(-/-) mice. In addition, inhibition by IC87114 of the major class IA PI3K catalytic subunit expressed in lymphocytes, p110delta, blocked transition of DN to DP cells in embryonic day 14.5 fetal thymic organ culture without affecting cell viability. In the absence of phosphatase and tensin homolog deleted on chromosome 10, where class IA PI3K signals would be amplified, the DN to DP transition was accelerated. In contrast, neither positive nor negative selection in Rag-2(-/-)TCR-transgenic mice was perturbed by the lack of p85alpha. These findings establish an important function of class IA PI3K in the pre-TCR-controlled developmental transition of DN to DP thymocytes.     

Role of phosphoinositide 3-kinase in monocyte recruitment under flow conditions

Chemokines such as the monocyte chemol attractant protein-1 (MCP-1) convert monocyte rolling to firm arrest under physiological flow conditions via integrin activation and simultaneously activate phosphoinositide 3-kinase (PI3K). Here we used adenoviral gene transfer and biochemical inhibitors to manipulate PI3K-dependent pathways in human monocytes. In in vitro lipid kinase assays from purified human monocytes, we showed that MCP-1 activates the "classical" PI3Kalpha pathway and not PI3Kgamma, a PI3K isoform thought to be activated only by the betagamma complex of heterotrimeric G proteins. The activity of PI3Kalpha in purified human monocytes was evident within 30 s. MCP-1-induced monocyte arrest was significantly inhibited both by wortmannin (n = 4; p < 0.01) and LY294002 (n = 4; p < 0.01) with restoration of the rolling phenotype (p < 0.05 for both inhibitors, compared with rolling of control monocytes after MCP-1 treatment). To test the hypothesis that activation of PI3K is sufficient to induce monocyte adhesion, we transduced the monocytic THP-1 cell line with a recombinant adenovirus (Ad) carrying a constitutively active mutant of PI3K (Ad.BD110). We examined the ability of these cells to adhere to human vascular endothelium (HUVEC) transduced with adenoviruses carrying E-selectin, intercellular adhesion molecule-1 (ICAM-1), and VCAM-1. Under flow conditions, ICAM-1- and VCAM-1-dependent firm adhesion of Ad.BD110-transduced THP-1 cells was enhanced compared with THP-1 cells infected with control Ad (n = 4; p < 0.01 for both). Adhesion augmented by constitutive PI3K activation was entirely abrogated by pretreatment with wortmannin (n = 3; p < 0.01). In contrast, a constitutively active Akt construct had no effect on THP-1 adhesion (n = 3; p = NS). We conclude that PI3K activation is necessary and sufficient to enhance monocytic adhesion under physiological flow conditions. BD110-expressing THP-1 cells should provide a useful tool for identifying the signaling pathways downstream of PI3K that are necessary for monocyte recruitment relevant to a variety of human vascular pathologies.     

A Role for phosphoinositide 3-kinase in the control of cell division and survival during retinal development

Neurogenesis in the retina requires the concerted action of three different cellular processes: proliferation, differentiation, and apoptosis. Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit. p110alpha has been shown to regulate cell division and survival. Little is known of its function in development, however, as p110alpha knockout mice exhibit CNS defects, but death at early embryonic stages impairs further study. Here, we examine the role of PI3K in mouse retina development by expressing an activating form of PI3K regulatory subunit, p65(PI3K), as a transgene in the retina. Mice expressing p65(PI3K) showed severely disrupted retina morphogenesis, with ectopic cell masses in the neuroepithelium that evolved into infoldings of adult retinal cell layers. These changes correlated with an altered cell proliferation/cell death balance at early developmental stages. Nonetheless, the most affected cell layer in adult retina was that of photoreceptors, which correlated with selectively increased survival of these cells at developmental stages at which cell division has ceased. These results demonstrate the relevance of accurate PI3K regulation for normal retinal development, supporting class IA PI3K involvement in induction of cell division at early stages of neurogenesis. These data also show that, even after cell division decline, PI3K activation mediates survival of differentiated neurons in vivo.     

Identification and characterization of the autophosphorylation sites of phosphoinositide 3-kinase isoforms beta and gamma

Class I phosphoinositide 3-kinases (PI3Ks) are bifunctional enzymes possessing lipid kinase activity and the capacity to phosphorylate their catalytic and/or regulatory subunits. In this study, in vitro autophosphorylation of the G protein-sensitive p85-coupled class I(A) PI3K beta and p101-coupled class I(B) PI3K gamma was examined. Autophosphorylation sites of both PI3K isoforms were mapped to C-terminal serine residues of the catalytic p110 subunit (i.e. serine 1070 of p110 beta and serine 1101 of p110 gamma). Like other class I(A) PI3K isoforms, autophosphorylation of p110 beta resulted in down-regulated PI3K beta lipid kinase activity. However, no inhibitory effect of p110 gamma autophosphorylation on PI3K gamma lipid kinase activity was observed. Moreover, PI3K beta and PI3K gamma differed in the regulation of their autophosphorylation. Whereas p110 beta autophosphorylation was stimulated neither by G beta gamma complexes nor by a phosphotyrosyl peptide derived from the platelet-derived growth factor receptor, autophosphorylation of p110 gamma was significantly enhanced by G beta gamma in a time- and concentration-dependent manner. In summary, we show that autophosphorylation of both PI3K beta and PI3K gamma occurs in a C-terminal region of the catalytic p110 subunit but differs in its regulation and possible functional consequences, suggesting distinct roles of autophosphorylation of PI3K beta and PI3K gamma.     

Role for phosphoinositide 3-kinase in Fc gamma RIIA-induced platelet shape change

Platelets transform from disks to irregular spheres, grow filopodia, form ruffles, and spread on surfaces coated with anti-FcRIIA antibody. FcRIIA cross-linking leads to a tenfold increase in actin filament barbed end exposure and robust actin assembly. Activation of the small GTPases Rac and Cdc42 follows FcRIIA cross-linking. Shape change, actin filament barbed end exposure, and quantifiable actin assembly require phosphoinositide 3-kinase (PI3-kinase) activity and a rise in intracellular calcium. PI3-kinase inhibition blocks activation of Rac, but not of Cdc42, and diminishes the association of Arp2/3 complex and CapZ with polymerized actin. Furthermore, addition of constitutively active D-3 phosphorylated polyphosphoinositides or recombinant PI3-kinase subunits to octylglucoside-permeabilized platelets elicits actin filament barbed end exposure by releasing gelsolin and CapZ from the cytoskeleton. Our findings place PI3-kinase activity upstream of Rac, gelsolin, and Arp2/3 complex activation induced by FcRIIA and clearly distinguish the FcRIIA signaling pathway to actin filament assembly from the thrombin receptor protease-activated receptor (PAR)-1 pathway. actin assembly; CD32A     

Role of PI3-kinase and PI4-kinase in actin polymerization during bovine sperm capacitation

We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP(2(4,5)), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 alpha regulatory subunit of PI3K) and PIKCB (PI4K beta) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 microM, respectively. PI4K activity and PIP(2(4,5)) production were blocked by 10 microM WT but not by 10 nM WT, whereas PI3K activity and PIP(3(3,4,5)) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP(2(4,5)) and F-actin formation. The increases in PIP(2(4,5)) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP(2(4,5)), PIP(3(3,4,5)), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP(2(4,5)) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP(3(3,4,5)) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.     

Cdc42 and phosphoinositide 3-kinase drive Rac-mediated actin polymerization downstream of c-Met in distinct and common pathways

Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise contributions of distinct Rho-GTPases, the phosphatidylinositol 3-kinases, and actin assembly regulators to c-Met-mediated actin reorganization are still elusive. Here we report that HGF-induced membrane ruffling and Listeria invasion mediated by the bacterial c-Met ligand internalin B (InlB) were significantly impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria invasion and membrane ruffling downstream of c-Met.     

The phosphoinositide 3-kinase pathway and cancer

Human tumours emerge as the result of multiple genetic and epigenetic aberrations that allow the proto-cancer cell to escape normal social control. Many signal transduction pathways become constitutively active during this process, and one whose importance is increasingly being appreciated involves phosphoinositide 3-kinase (PI3-kinase). This pathway normally regulates important cell decisions such as growth, division, survival and migration, and when deregulated it can confer malignant potential to the ensuing tumour. However, constitutive activation of the PI3-kinase pathway might provide attractive therapeutic targets for the design of small-molecule inhibitors. This review discusses events upstream and downstream of PI3-kinase activity in the PI3-kinase signalling pathway, how PI3-kinase is deregulated in human tumourigenesis, and how this is being targeted clinically.     

Roles of non-catalytic subunits in gbetagamma-induced activation of class I phosphoinositide 3-kinase isoforms beta and gamma.

By using purified preparations we show that nanomolar concentrations of Gbetagamma significantly stimulated lipid kinase activity of phosphatidylinositol 3-kinase (PI3K) beta and PI3Kgamma in the presence as well as in the absence of non-catalytic subunits such as p85alpha or p101. Concomitantly, Gbetagamma stimulated autophosphorylation of the catalytic subunit of PI3Kgamma (EC(50), 30 nM; stoichiometry >/=0.6 mol of P(i)/mol of p110gamma), which also occurred in the absence of p101. Surprisingly, we found that p101 affected the lipid substrate preference of PI3Kgamma in its Gbetagamma-stimulated state. With phosphatidylinositol as substrate, p110gamma but not p101/p110gamma was significantly stimulated by Gbetagamma to form PI-3-phosphate (EC(50), 20 nM). The opposite situation was found when PI-4,5-bisphosphate served as substrate. Gbetagamma efficiently and potently (EC(50), 5 nM) activated the p101/p110gamma heterodimer but negligibly stimulated the p110gamma monomer to form PI-3,4,5-trisphosphate. However, this weak stimulatory effect on p110gamma was overcome by excess concentrations of Gbetagamma (EC(50), 100 nM). This finding is in accordance with the in vivo situation, where activated PI3K catalyzes the formation of PI-3,4,5-trisphosphate but not PI-3-phosphate. We conclude that p101 is responsible for PI-4, 5-bisphosphate substrate selectivity of PI3Kgamma by sensitizing p110gamma toward Gbetagamma in the presence of PI-4,5-P(2).     

Assigning functional domains within the p101 regulatory subunit of phosphoinositide 3-kinase gamma

Phosphoinositide 3-Kinase (PI3K) gamma is a lipid kinase that is regulated by G-protein-coupled receptors. It plays a crucial role in inflammatory and allergic processes. Activation of PI3Kgamma is primarily mediated by Gbetagamma subunits. The regulatory p101 subunit of PI3Kgamma binds to Gbetagamma and, thereby, recruits the catalytic p110gamma subunit to the plasma membrane. Despite its crucial role in the activation of PI3Kgamma, the structural organization of p101 is still largely elusive. Employing fluorescence resonance energy transfer measurements, coimmunoprecipitation and colocalization studies with p101 deletion mutants, we show here that distinct regions within the p101 primary structure are responsible for interaction with p110gamma and Gbetagamma. The p110gamma binding site is confined to the N terminus, whereas binding to Gbetagamma is mediated by a C-terminal domain of p101. These domains appear to be highly conserved among various species ranging from Xenopus to men. In addition to establishing a domain structure for p101, our results point to the existence of a previously unknown, p101-related regulatory subunit for PI3Kgamma.     

Cloning, expression, purification, and characterization of the human Class Ia phosphoinositide 3-kinase isoforms

The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct. To further study their unique biochemical properties, the three human Class Ia PI3K (alpha, beta, and delta) p110 catalytic domains were cloned and co-expressed with the p85alpha regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85alpha. Successful expression and purification of each p85alpha/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85alpha/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24mg/L cell culture media for p85alpha/p110alpha, 17.5mg/L for p85alpha/p110delta, and 3.5mg/L for p85alpha/p110beta. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The activities of the three p85alpha/p110 proteins and the Class Ib p110gamma catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome. All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The surface concentrations that gave maximal activity for each human isoform with 0.5mM PIP2 were 2.5mol% PIP2 for p110gamma, 7.5mol% for p85alpha/p110beta, and 10mol% PIP2 for p85alpha/p110alpha and p85alpha/p110delta. The specific activity of p85alpha/p110alpha was three to five times higher than that of the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells.     

Role of phosphoinositide 3-kinase in the autophagic death of serum-deprived PC12 cells

The death of serum-deprived undifferentiated PC12 cells shows both autophagic and apoptotic features. Since it is still controversial whether the autophagy is instrumental in the cell death or a mere epiphenomenon, we tested the effects of inhibiting the autophagy by a variety of phosphoinositide 3-kinase inhibitors, and provided evidence that the autophagy, or a related trafficking event, is indeed instrumental in the cell death. Furthermore, by comparing the effects of PI3-K inhibition and caspase-inhibition on autophagic and apoptotic cellular events, we showed that in this case the autophagic and apoptotic mechanisms mediate cell death by parallel pathways and do not act in series.Financial support: grants 31-50598.97 and 31-61736.00 from the Swiss National Science Foundation     

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase.

p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85alpha-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.     

Mechanism of constitutive phosphoinositide 3-kinase activation by oncogenic mutants of the p85 regulatory subunit

p85/p110 phosphoinositide 3-kinases regulate multiple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhibits the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the conformationally rigid iSH2 domain tethers p110 to p85, facilitating regulatory interactions between p110 and the p85 nSH2 domain. In an oncogenic mutant of murine p85, truncation at residue 571 leads to constitutively increased phosphoinositide 3-kinase activity, which has been proposed to result from either loss of an inhibitory Ser-608 autophosphorylation site or altered interactions with cellular regulatory factors. We have examined this mutant (referred to as p65) in vitro and find that p65 binds but does not inhibit p110, leading to constitutive p110 activity. This activated phenotype is observed with recombinant proteins in the absence of cellular factors. Importantly, this effect is also produced by truncating p85ni at residue 571. Thus, the phenotype is not because of loss of the Ser-608 inhibitory autophosphorylation site, which is not present in p85ni. To determine the structural basis for the phenotype of p65, we used a broadly applicable spin label/NMR approach to define the positioning of the nSH2 domain relative to the iSH2 domain. We found that one face of the nSH2 domain packs against the 581-593 region of the iSH2 domain. The loss of this interaction in the truncated p65 would remove the orienting constraints on the nSH2 domain, leading to a loss of p110 regulation by the nSH2. Based on these findings, we propose a general model for oncogenic mutants of p85 and p110 in which disruption of nSH2-p110 regulatory contacts leads to constitutive p110 activity.     

The p85alpha regulatory subunit of phosphoinositide 3-kinase potentiates c-Jun N-terminal kinase-mediated insulin resistance

Insulin resistance is a defining feature of type 2 diabetes and the metabolic syndrome. While the molecular mechanisms of insulin resistance are multiple, recent evidence suggests that attenuation of insulin signaling by c-Jun N-terminal kinase (JNK) may be a central part of the pathobiology of insulin resistance. Here we demonstrate that the p85alpha regulatory subunit of phosphoinositide 3-kinase (PI3K), a key mediator of insulin's metabolic actions, is also required for the activation of JNK in states of insulin resistance, including high-fat diet-induced obesity and JNK1 overexpression. The requirement of the p85alpha regulatory subunit for JNK occurs independently of its role as a component of the PI3K heterodimer and occurs only in response to specific stimuli, namely, insulin and tunicamycin, a chemical that induces endoplasmic reticulum stress. We further show that insulin and p85 activate JNK by via cdc42 and MKK4. The activation of this cdc42/JNK pathway requires both an intact N terminus and functional SH2 domains within the C terminus of the p85alpha regulatory subunit. Thus, p85alpha plays a dual role in regulating insulin sensitivity and may mediate cross talk between the PI3K and stress kinase pathways.     

The p85 regulatory subunit controls sequential activation of phosphoinositide 3-kinase by Tyr kinases and Ras

Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit that regulates a variety of cell responses, including cell division and survival. PI3K is activated following Tyr kinase stimulation and by Ras. We found that the C-terminal region of p85, including the C-Src homology 2 (C-SH2) domain and part of the inter-SH2 region, protects the p110 catalytic subunit from Ras-induced activation. Although the p110 activity associated with a C-terminal p85 deletion mutant increased significantly in the presence of an active form of Ras, purified wild type p85-p110 was only slightly stimulated by active Ras. Nonetheless, incubation of purified p85-p110 with Tyr-phosphorylated peptides, which mimic the activated platelet-derived growth factor receptor, restored Ras-induced p85-p110 activation. In conclusion, p85 inhibits p110 activation by Ras; this blockage is released by Tyr kinase stimulation, showing that the classical mechanism of class IA PI3K stimulation mediated by Tyr kinases also regulates Ras-induced PI3K activation.