Regulation of class IA PI3Ks: is there a role for monomeric PI3K subunits?

Class IA PI3Ks (phosphoinositide 3-kinases) consist of a p110 catalytic subunit bound to one of five regulatory subunits, known as p85s. Under unstimulated conditions, p85 stabilizes the labile p110 protein, while inhibiting its catalytic activity. Recruitment of the p85-p110 complex to receptors and adaptor proteins via the p85 SH2 (Src homology 2) domains alleviates this inhibition, leading to PI3K activation and production of PIP(3) (phosphatidylinositol 3,4,5-trisphosphate). Four independent p85 KO (knockout) mouse lines have been generated. Remarkably, PI3K signalling in insulin-sensitive tissues of these mice is increased. The existence of p110-free p85 in insulin-responsive cells has been invoked to explain this observation. Such a monomeric p85 would compete with heterodimeric p85-p110 for pTyr (phosphotyrosine) recruitment, and thus repress PI3K activity. Reduction in the pool of p110-free p85 in p85 KO mice was thought to allow recruitment of functional heterodimeric p85-p110, leading to increased PI3K activity. However, recent results indicate that monomeric p85, like p110, is unstable in cells. Moreover, overexpressed free p85 does not necessarily compete with heterodimeric p85-p110 for receptor binding. Using a variety of approaches, we have observed a 1:1 ratio between the p85 and p110 subunits in murine cell lines and primary tissues. Alternative models to explain the increase in PI3K signalling in insulin-responsive cells of p85 KO mice, based on possible effects of p85 deletion on phosphatases acting on PIP(3), are discussed.

A selective inhibitor of the p110delta isoform of PI 3-kinase inhibits AML cell proliferation and survival and increases the cytotoxic effects of VP16.

Current therapy for acute myeloid leukaemia (AML) is suboptimal with a high incidence of relapse. There is strong evidence that constitutive phosphoinositide 3-kinase (PI3K) activity plays a significant role in the pathophysiology of AML. PI3K products are derived from the activity of a number of PI3K catalytic isoforms (class I, II and III) but the relative contribution of these enzymes in AML remains unknown. As non-isoform-selective inhibitors of PI3K such as LY294002 may produce unwanted toxicity to normal tissues, we have investigated the role of the leukocyte-restricted p110delta PI3K isoform in 14 cases of AML. p110delta was detected in all cases whereas the expression levels of the other class I PI3Ks varied more widely, and were often undetectable. The p110delta-selective compound IC87114 inhibited constitutive phosphorylation of the PI3K target Akt/PKB and reduced cell number to a mean of 66+/-5% (range 14-88%). In eight cases, the combination of IC87114 and VP16 (a topoisomerase II inhibitor) was synergistic in reducing viable cell number, and was associated with a reduction in constitutive NF-kappaB activity. IC87114 did not have direct adverse effects or enhance the activity of VP16 on the proliferation and survival of normal haemopoietic progenitors. Overall, our results identify the p110delta isoform as a potential therapeutic target in AML and support a clinical approach to use isoform-selective over broad-spectrum PI3K inhibitors.

Phosphoinositide 3-kinase in T cell activation and survival.

PI3Ks (phosphoinositide 3-kinases) regulate diverse signalling pathways involved in growth, proliferation, survival, differentiation and metabolism. In T cells, PI3Ks can be activated by a number of different receptors, including the TcR (T cell receptor), co-stimulatory receptors, cytokine receptors and chemokine receptors. However, the specific roles of PI3Ks downstream of these receptors vary. An inactivating mutation in the leucocyte-specific PI3K isoform p110delta results in impaired TcR-dependent proliferation under circumstances where CD28 co-stimulation is blocked or not required. Recruitment and activation of PI3K by CD28 promotes survival by inducing increased expression of Bcl-X(L). However, CD28 engages additional signals that regulate proliferation and interleukin-2 production independently of PI3K. Thus a model emerges whereby PI3K is involved in both TcR and CD28 signalling, but each receptor may only exploit a subset of the signalling pathways potentially controlled by PI3K activation.

PI3K-signalling in B- and T-cells: insights from gene-targeted mice.

Phosphoinositide 3-kinases (PI3Ks) are important signalling enzymes in most cell types. Recent gene targeting studies have shed light on the importance of this family of lipid kinases in the immune system, and the complex mechanisms by which these kinases are regulated in vivo. We have recently reported a phenotype of mice in which the p110 delta PI3K catalytic subunit was inactivated by point mutation. In the present paper, we compare and contrast the phenotypes of p110 delta mutant mice with those of mice that lack p85 alpha or p110 gamma, and discuss these in the context of PI3K signalling in B- and T-cells.

New responsibilities for the PI3K regulatory subunit p85 alpha.

Class IA phosphoinositide-3 kinases (PI3Ks) are heterodimeric enzymes that regulate many signal transduction pathways. The p85 regulatory subunit recruits the p110 catalytic subunit to the membrane, where p110 phosphorylates inositol lipids. Recent studies present evidence for an additional role for p85alpha in the regulation of actin cytoskeleton. Okkenhaug and Vanhaesebroeck discuss these results and ask whether experiments describing p85alpha knockout mice need to be reinterpreted.

Synthesis and function of 3-phosphorylated inositol lipids.

The 3-phosphorylated inositol lipids fulfill roles as second messengers by interacting with the lipid binding domains of a variety of cellular proteins. Such interactions can affect the subcellular localization and aggregation of target proteins, and through allosteric effects, their activity. Generation of 3-phosphoinositides has been documented to influence diverse cellular pathways and hence alter a spectrum of fundamental cellular activities. This review is focused on the 3-phosphoinositide lipids, the synthesis of which is acutely triggered by extracellular stimuli, the enzymes responsible for their synthesis and metabolism, and their cell biological roles. Much knowledge has recently been gained through structural insights into the lipid kinases, their interaction with inhibitors, and the way their 3-phosphoinositide products interact with protein targets. This field is now moving toward a genetic dissection of 3-phosphoinositide action in a variety of model organisms. Such approaches will reveal the true role of the 3-phosphoinositides at the organismal level in health and disease.

Distinct mechanisms of alpha 5beta 1 integrin activation by Ha-Ras and R-Ras.

To investigate the possible roles of the Ras/Rho family members in the inside-out signals to activate integrins, we examined the ability of Ras/Rho small GTPases to stimulate avidity of alpha(5)beta(1) (VLA-5) to fibronectin in bone marrow-derived mast cells. We found that both Ha-Ras(Val-12) and R-Ras(Val-38) had strong stimulatory effects on adhesion and ligand binding activity of VLA-5 to fibronectin. However, only Ha-Ras(Val-12)-, but not R-Ras(Val-38)-induced adhesion was inhibited by wortmannin, which suggests that Ha-Ras(Val-12) is dependent on phosphatidylinositol (PI) 3-kinase on adhesion whereas R-Ras(Val-38) has another PI 3-kinase independent pathway to induce adhesion. The effector loop mutant Ha-Ras(Val-12)E37G, but not Y40C retained the ability to stimulate adhesion of mast cells to fibronectin. Consistently, PI 3-kinase p110delta, predominantly expressed in mast cells, interacted with Ha-Ras(Val-12) E37G, but not Y40C, which was also correlated with the levels of Akt phosphorylation in mast cells. Furthermore, marked adhesion was induced by a membrane-targeted version of p110delta. These results indicate that Ha-Ras(Val-12) activated VLA-5 through PI 3-kinase p110delta. The mutational effects of the R-Ras effector loop region on adhesion were not correlated with PI 3-kinase activities, consistent with our contention that R-Ras has a distinct pathway to modulate avidity of VLA-5.

The PI3K-PDK1 connection: more than just a road to PKB.

Phosphoinositide 3-kinases (PI3Ks) generate specific inositol lipids that have been implicated in the regulation of cell growth, proliferation, survival, differentiation and cytoskeletal changes. One of the best characterized targets of PI3K lipid products is the protein kinase Akt or protein kinase B (PKB). In quiescent cells, PKB resides in the cytosol in a low-activity conformation. Upon cellular stimulation, PKB is activated through recruitment to cellular membranes by PI3K lipid products and phosphorylation by 3'-phosphoinositide-dependent kinase-1 (PDK1). Here we review the mechanism by which PKB is activated and the downstream actions of this multifunctional kinase. We also discuss the evidence that PDK1 may be involved in the activation of protein kinases other than PKB, the mechanisms by which this activity of PDK1 could be regulated and the possibility that some of the currently postulated PKB substrates targets might in fact be phosphorylated by PDK1-regulated kinases other than PKB.

The PI 3-kinase isoforms p110(alpha) and p110(beta) have differential roles in PDGF- and insulin-mediated signaling.

Phosphoinositide 3'-kinases constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. Phosphoinositide 3'-kinases that bind to the platelet-derived growth factor receptor are composed of two subunits: the p85 subunit acts as an adapter and couples the catalytic p110 subunit to the activated receptor. There are different isoforms of p85 as well as of p110, the individual roles of which have been elusive. Using microinjection of inhibitory antibodies specific for either p110(alpha) or p110(beta) we have investigated the involvement of the two p110 isoforms in platelet-derived growth factor- and insulin-induced actin reorganization in porcine aortic endothelial cells. We have found that antibodies against p110(alpha), but not antibodies against p110(beta), inhibit platelet-derived growth factor-stimulated actin reorganization, whereas the reverse is true for inhibition of insulin-induced actin reorganization. These data indicate that the two phosphoinositide 3'-kinase isoforms have distinct roles in signal transduction pathways induced by platelet-derived growth factor and insulin.

Signaling by distinct classes of phosphoinositide 3-kinases.

Many signaling pathways converge on and regulate phosphoinositide 3-kinase (PI3K) enzymes whose inositol lipid products are key mediators of intracellular signaling. Different PI3K isoforms generate specific lipids that bind to FYVE and pleckstrin homology (PH) domains in a variety of proteins, affecting their localization, conformation, and activities. Here we review the activation mechanisms of the different types of PI3Ks and their downstream actions, with focus on the PI3Ks that are acutely triggered by extracellular stimulation.

Signalling through phosphoinositide 3-kinases: the lipids take centre stage.

Phosphoinositide 3-kinases (PI3Ks) phosphorylate inositol lipids at the 3' position of the inositol ring to generate the 3-phosphoinositides PI(3)P, PI(3,4) P2 and PI(3,4,5) P3. Recent research has shown that one way in which these lipids function in signal transduction and membrane trafficking is by interacting with 3-phosphoinositide-binding modules in a broad variety of proteins. Specifically, certain FYVE domains bind PI(3)P whereas certain pleckstrin homology domains bind PI(3,4) P2 and/or PI(3,4,5) P3. Also in 1998, PTEN - a major tumour suppressor in human cancer - was also shown to antagonise PI3K signalling by removing the 3-phosphate from 3-phosphoinositides.

Autophosphorylation of p110delta phosphoinositide 3-kinase: a new paradigm for the regulation of lipid kinases in vitro and in vivo.

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases which also possess an in vitro protein kinase activity towards themselves or their adaptor proteins. The physiological relevance of these phosphorylations is unclear at present. Here, the protein kinase activity of the tyrosine kinase-linked PI3K, p110delta, is characterized and its functional impact assessed. In vitro autophosphorylation of p110delta completely down-regulates its lipid kinase activity. The single site of autophosphorylation was mapped to Ser1039 at the C-terminus of p110delta. Antisera specific for phospho-Ser1039 revealed a very low level of phosphorylation of this residue in cell lines. However, p110delta that is recruited to activated receptors (such as CD28 in T cells) shows a time-dependent increase in Ser1039 phosphorylation and a concomitant decrease in associated lipid kinase activity. Treatment of cells with okadaic acid, an inhibitor of Ser/Thr phosphatases, also dramatically increases the level of Ser1039-phosphorylated p110delta. LY294002 and wortmannin blocked these in vivo increases in Ser1039 phosphorylation, consistent with the notion that PI3Ks, and possibly p110delta itself, are involved in the in vivo phosphorylation of p110delta. In summary, we show that PI3Ks are subject to regulatory phosphorylations in vivo similar to those identified under in vitro conditions, identifying a new level of control of these signalling molecules.

Autocrine tumor necrosis factor (TNF) and lymphotoxin (LT) alpha differentially modulate cellular sensitivity to TNF/LT-alpha cytotoxicity in L929 cells.

Tumor necrosis factor (TNF) and lymphotoxin (LT) alpha are structurally and functionally related cytokines. We expressed the TNF and LT-alpha genes in murine fibrosarcoma L929r2 cells, which can be sensitized to TNF/LT-alpha-dependent necrosis by inhibitors of transcription or translation. Autocrine production of murine TNF in L929r2 cells completely downmodulated the expression of the 55- and 75-kD TNF receptors, resulting in resistance to TNF/LT-alpha cytotoxicity. Partial downmodulation of the 55-kD receptor was observed in human TNF-producing L929r2 cells. In contrast, an unaltered TNF receptor expression was found on LT-alpha L929r2 transfectants. Hence, although similar cytotoxic effects are induced by extracellularly administered TNF and LT-alpha, endogenous expression of these cytokines fundamentally differs in the way they modulate TNF receptor expression. Unlike LT-alpha, secreted by the classical pathway, TNF is first formed as a membrane-bound protein, which is responsible for receptor downmodulation. To explore whether the different pathways for secretion of TNF and LT-alpha explain this difference, we examined the effect of membrane-bound LT-alpha expression. This was obtained by exchange of the classical signal sequence of LT-alpha for the membrane anchor of chicken hepatic lectin. Membrane retention of LT-alpha resulted indeed in receptor downmodulation and TNF/LT-alpha resistance. We conclude that membrane retention of newly synthesized TNF or LT-alpha is absolutely required for receptor downmodulation and TNF/LT-alpha resistance.

A redox-triggered ras-effector interaction. Recruitment of phosphatidylinositol 3'-kinase to Ras by redox stress.

Reactive free radical species are known to trigger biochemical events culminating in transcription factor activation and modulation of gene expression. The cytosolic signaling events triggered by free radicals that result in nuclear responses are largely unknown. Here we identify a signaling cascade triggered immediately upon redox activation of Ras. We examined two physiologically relevant models of redox signaling: 1) nitric oxide in human T cells, and 2) advanced glycation end product in rat pheochromocytoma cells. Reactive free radical species generated by nitric oxide donors and the interaction of advanced glycation end product with its receptor led to the recruitment of p85/p110 phosphatidylinositol 3'-kinase (PI3K) to the plasma membrane, where it associated directly with the effector domain of Ras and became activated. Only the p110beta and p110delta (but not p110alpha) catalytic subunits were recruited by redox-activated Ras. Activation of downstream targets of PI3K such as protein kinase B/Akt and mitogen-activated protein kinase was found to be PI3K dependent. Our study demonstrates that nitrosative and oxidative stressors trigger Ras-dependent and PI3K-regulated events in cells and define a biochemical pathway that is triggered by redox signaling.

Induction of unresponsiveness to tumor necrosis factor (TNF) after autocrine TNF expression requires TNF membrane retention.

Tumor necrosis factor (TNF) has a specific gene-inducing activity on many cell types and exerts a cytotoxic effect on a number of tumor cell lines. However, several tumor cell types are resistant to TNF-induced effects, and some of these produce TNF. We previously demonstrated that introduction of an exogenous TNF gene in the TNF-sensitive cell line L929sA induced autocrine TNF production and unresponsiveness to the cytotoxic activity of TNF. This resistance required biologically active TNF and was correlated with complete down-modulation of the TNF receptors on the cell surface. We have now characterized this process in more detail. The role of expression of the membrane-bound TNF proform and its subsequent proteolytic processing in the induction of TNF unresponsiveness was investigated. Exchange of the TNF presequence for the signal sequence of interleukin-6 resulted in production of secreted TNF, but not in induction of TNF resistance. On the other hand, expression of non-secretable, membrane-bound TNF generated complete TNF unresponsiveness. To explore whether the requirement for anchoring reflected a specific functional role of the TNF presequence, the latter was replaced by the membrane anchor of trimeric chicken hepatic lectin. Expression of this construct induced complete TNF unresponsiveness. Hence, the role of the TNF presequence in the induction of TNF unresponsiveness only involves its function as a membrane anchor, which permits oligomerization of the TNF molecule into a biologically active homotrimer.

Phosphoinositide 3-kinases: a conserved family of signal transducers.

Phosphoinositide 3-kinases (PI3Ks) generate lipids that are implicated in receptor-stimulated signalling and in the regulation of membrane traffic. Several distinct classes of PI3Ks have now been identified that have been conserved throughout eukaryotic evolution. Potential signalling pathways downstream of PI3Ks have been elucidated and PI3K function is now being characterised in several model organisms.

P110delta, a novel phosphoinositide 3-kinase in leukocytes.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been implicated in signal transduction through tyrosine kinase- and heterotrimeric G-protein-linked receptors. We report herein the cloning and characterization of p110delta, a novel class I PI3K. Like p110alpha and p110beta, other class I PI3Ks, p110delta displays a broad phosphoinositide lipid substrate specificity and interacts with SH2/SH3 domain-containing p85 adaptor proteins and with GTP-bound Ras. In contrast to the widely distributed p110alpha and beta, p110delta is exclusively found in leukocytes. In these cells, p110alpha and delta both associate with the p85alpha and beta adaptor subunits and are similarly recruited to activated signaling complexes after treatment with the cytokines interleukin 3 and 4 and stem cell factor. Thus, these class I PI3Ks appear not to be distinguishable at the level of p85 adaptor selection or recruitment to activated receptor complexes. However, distinct biochemical and structural features of p110delta suggest divergent functional/regulatory capacities for this PI3K. Unlike p110alpha, p110delta does not phosphorylate p85 but instead harbors an intrinsic autophosphorylation capacity. In addition, the p110delta catalytic domain contains unique potential protein-protein interaction modules such as a Pro-rich region and a basic-region leucine-zipper (bZIP)-like domain. Possible selective functions of p110delta in white blood cells are discussed.

Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation.

We have reported previously that Ras interacts with the catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in a GTP-dependent manner. The affinity of the interaction of Ras-GTP with p85alpha/p110alpha is shown here to be approximately 150 nM. The site of interaction on the p110alpha and beta isoforms of PI 3-kinase lies between amino acid residues 133 and 314. A point mutation in this region, K227E, blocks the GTP-dependent interaction of PI 3-kinase p110alpha with Ras in vitro and the ability of Ras to activate PI 3-kinase in intact cells. In addition, this mutation elevates the basal activity of PI 3-kinase in intact cells, suggesting a direct influence of the Ras binding site on the catalytic activity of PI 3-kinase. Using an in vitro reconstitution assay, it is shown that the interaction of Ras-GTP, but not Ras-GDP, with PI 3-kinase leads to an increase in its enzymatic activity. This stimulation is synergistic with the effect of tyrosine phosphopeptide binding to p85, particularly at suboptimal peptide concentrations. These data show that PI 3-kinase is regulated by a number of mechanisms, and that Ras contributes to the activation of this lipid kinase synergistically with tyrosine kinases.

Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction.

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.