PI3-kinase inhibition: a target for drug development?

The phosphoinositide 3-kinases (PI3-kinases) are a ubiquitously expressed enzyme family that, through the generation of phospholipid second messengers, play a key role in the regulation of many cellular processes. These include motility, proliferation and survival, and carbohydrate metabolism. Members of the PI3-kinase family and related kinases, their mechanism of activation and the cellular events that they influence are described in this review. As knowledge of their involvement in disease processes increases, the PI3-kinases appear to be an increasingly attractive target for drug development, particularly in the fields of cancer and other proliferative diseases, and in the treatment of inflammatory and immunological conditions. Evidence of the functional specialization of PI3-kinase isoforms suggests that selective inhibition with acceptable toxicity might be possible.     

Intracellular trafficking and turnover of phosphatidylinositol 3-phosphate

Phosphatidylinositol 3-kinases (PI 3-kinases) regulate cellular functions through the 3'-phosphorylation of phosphatidylinositol (PI) and its derivatives. The PI 3-kinase product phosphatidylinositol 3-phosphate [PI(3)P] functions to recruit and activate effector proteins containing FYVE zinc finger domains. These proteins have various functions in endocytic membrane trafficking, cytoskeletal regulation and signal transduction. In order to understand the function of FYVE proteins, it is essential to study the formation, localisation, trafficking and turnover of PI(3)P. Here we review recent evidence that PI(3)P is formed on early endosomes through the activity of a PI 3-kinase which is recruited by the GTPase Rab5, and that the PI(3)P is subsequently internalised into intralumenal vesicles of multivesicular endosomes for turnover.     

Phosphatidylinositol 4-kinase IIIbeta regulates the transport of ceramide between the endoplasmic reticulum and Golgi

The recently identified ceramide transfer protein, CERT, is responsible for the bulk of ceramide transport from the endoplasmic reticulum (ER) to the Golgi. CERT has a C-terminal START domain for ceramide binding and an N-terminal pleck-strin homology domain that binds phosphatidylinositol 4-phosphate suggesting that phosphatidylinositol (PI) 4-kinases are involved in the regulation of CERT-mediated ceramide transport. In the present study fluorescent analogues were used to follow the ER to Golgi transport of ceramide to determine which of the four mammalian PI 4-kinases are involved in this process. Overexpression of pleckstrin homology domains that bind phosphatidylinositol 4-phosphate strongly inhibited the transport of C5-BODIPY-ceramide to the Golgi. A newly identified PI 3-kinase inhibitor, PIK93 that selectively inhibits the type III PI 4-kinase beta enzyme, and small interfering RNA-mediated down-regulation of the individual PI 4-kinase enzymes, revealed that PI 4-kinase beta has a dominant role in ceramide transport between the ER and Golgi. Accordingly, inhibition of PI 4-kinase III beta either by wortmannin or PIK93 inhibited the conversion of [3H]serine-labeled endogenous ceramide to sphingomyelin. Therefore, PI 4-kinase beta is a key enzyme in the control of spingomyelin synthesis by controlling the flow of ceramide from the ER to the Golgi compartment.     

Phosphoinositide 3-kinases and membrane traffic

Phosphoinositide 3-kinases (PI 3-kinases) and their 3-phosphoinositide products were identified initially as components of intracellular signalling pathways emanating from cell surface receptors. A new role for 3-phosphoinositides in the constitutive movement o f proteins from one intracellular compartment to another was proposed with the discovery of homology between the product of a yeast gene important for vacuolar sorting, Vps34p, and a mammalian PI 3-kinase. Recent studies have implicated PI 3-kinase as an essential component in membrane traffic at specific steps o f the trans-Golgi-network-endosomal pre-lysosomal system. Evidence largely emerging from the insulin-stimulated glucose transport system suggests that PI 3-kinase may also mediate the effects o f growth factors on membrane traffic events. These studies suggest a possible link between growth-factor-stimulated and constitutive membrane traffic in the endosomal system.     

eIF4E binding protein 1 and H-Ras are novel substrates for the protein kinase activity of class-I phosphoinositide 3-kinase

Class-I phosphoinositide 3-kinases (PI 3-kinases) are dual specificity enzymes that possess both lipid and protein kinase activity. While the best characterized property of this protein kinase is as an autokinase activity, there have also been reports it can phosphorylate exogenous substrates including peptides, IRS-1 and PDE-3B. The identification of two novel potential protein substrates of PI 3-kinase is described here. By employing in vitro kinase assays using recombinant proteins as the substrates, it is shown that the translational regulator 4EBP1 becomes phosphorylated by the p110alpha and p110gamma isoforms of class-I PI 3-kinases. The lipid kinase activity of both these isoforms is increased by allosteric binding of H-Ras or betagamma subunits of heterotrimeric G proteins, but we find this is not the case for the protein kinase activity. Surprisingly though, a site on H-Ras is phosphorylated by p110alpha and p110gamma. This raises the possibility that these proteins could serve as physiological substrates for the protein kinase activity of PI 3-kinase and suggests this activity operates in a physiological context by phosphorylating substrates other than the PI 3-kinase itself. This may be particularly important in regulating the interaction of Ras with PI 3-kinase.     

Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3'-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.     

Direct interaction of major histocompatibility complex class II-derived peptides with class Ia phosphoinositide 3-kinase results in dose-dependent stimulatory effects

Peptides corresponding to residues 65-79 of human lymphocyte antigen class II sequence (DQA*03011) are cell-permeable and at high concentrations block activation of protein kinase B/Akt and p70-S6 kinase in T-cells, effects attributed to inhibition of phosphoinositide (PI) 3-kinase activity. To understand the molecular basis of this, we analyzed the effect this peptide had on activity of class I PI 3-kinases. Although there was no effect on the activity of class Ib PI 3-kinase or on the protein kinase activity of class I PI 3-kinases, there was a biphasic effect on lipid kinase activity of the class Ia enzymes. There was an inhibition of activity at higher peptide concentrations because of a formation of insoluble complexes between peptide and enzyme. Conversely, at lower peptide concentrations there was a profound activation of PI 3-kinase activity of class Ia PI 3-kinases. Studies of peptide variants revealed that all active peptides conform to heptad repeat motifs characteristic of coiled-coil helices. Surface plasmon resonance studies confirmed direct sequence-specific binding of active peptide to the p85alpha adapter subunit of class Ia PI 3-kinase. Active peptides also activated protein kinase B and extracellular signal-regulated kinase (ERK) in vivo in a wortmannin-sensitive manner while reducing recoverable cellular p85 levels. These results indicate that the human lymphocyte antigen class II-derived peptides regulate PI 3-kinase by direct interaction, probably via the coiled-coil domain. These peptides define a novel mechanism of regulating PI 3-kinase and will provide a useful tool for specifically dissecting the function of class Ia PI 3-kinase in cells and for probing structure-function relationships in the class Ia PI 3-kinase heterodimers.     

Identification of a unique co-operative phosphoinositide 3-kinase signaling mechanism regulating integrin alpha IIb beta 3 adhesive function in platelets

Phosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (p110 beta and p110 gamma) have been implicated in G(i)-dependent integrin alpha(IIb)beta(3) regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110 beta is primarily responsible for G(i)-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin alpha(IIb)beta(3) activation in p110 gamma(-/-) platelets was not associated with alterations in the levels of PI(3,4)P(2) or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that p110 gamma principally regulated integrin alpha(IIb)beta(3) activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110 gamma, led to a major reduction in integrin alpha(IIb)beta(3) activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110 gamma in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110 beta and p110 gamma is critical for P2Y(12)/G(i)-dependent integrin alpha(IIb)beta(3) regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways.     

Activation of host cell phosphatidylinositol 3-kinases by Trypanosoma cruzi infection

Trypanosoma cruzi, the causative agent of Chagas' disease in humans, is an intracellular protozoan parasite with the ability to invade a wide variety of mammalian cells by a unique and remarkable process in cell biology that is poorly understood. Here we present evidence suggesting a role for the host phosphatidylinositol (PI) 3-kinases during T. cruzi invasion. The PI 3-kinase inhibitor wortmannin marked inhibited T. cruzi infection when macrophages were pretreated for 20 min at 37 degrees C before inoculation. Infection of macrophages with T. cruzi markedly stimulated the formation of the lipid products of the phosphatidylinositol (PI) 3-kinases, PI 3-phospate, PI 3,4-biphosphate, and PI 3,4,5-triphosphate, but not PI 4-phosphate or PI 4,5-biphosphate. This activation was inhibited by wortmannin. Infection with T. cruzi also stimulated a marked increase in the in vitro lipid kinase activities that are present in the immunoprecipitates of anti-p85 subunit of class I PI 3-kinase and anti-phosphotyrosine. In addition, T. cruzi invasion also activated lipid kinase activity found in immunoprecipitates of class II and class III PI 3-kinases. These data demonstrate that T. cruzi invasion into macrophages strongly activates separated PI 3-kinase isoforms. Furthermore, the inhibition of the class I and class III PI 3-kinase activities abolishes the parasite entry into macrophages. These findings suggest a prominent role for the host PI 3-kinase activities during the T. cruzi infection process.     

Anionic phospholipids are involved in membrane targeting of PI 3-kinase gamma

Phosphoinositide 3-kinases (PI 3-kinases) have critical roles in diverse cellular signaling processes and in protein trafficking. In contrast to the class I PI 3-kinases alpha, beta, and delta which bind via src homology 2 (SH2) domains of adaptor proteins to tyrosine kinase receptors, the mechanism of recruitment of the PI 3-kinase gamma to membranes is unknown. We report in vitro experiments using immobilized proteins and small unilamellar vesicles which suggest an involvement of anionic phospholipids in membrane association of PI 3-kinase gamma. Furthermore we provide evidence that the enzyme displays beside the catalytic center a phospholipid binding domain which is essential for enzyme activity.     

A high-throughput liposome substrate assay with automated lipid extraction process for PI 3-kinase

The signaling pathways involving lipid kinase class I phosphatidylinositol 3-kinases (PI 3-kinases) regulate cell growth, proliferation, and survival. Class I PI 3-kinases catalyze the conversion of PI (4,5)P(2) to PI (3,4,5)P(3), which acts as a lipid second messenger to activate mitogenic signaling cascades. Recently, p110alpha, a class IA PI 3-kinase, was found to be mutated frequently in many human cancers. Therefore, it is increasingly studied as an anticancer drug target. Traditionally, PI 3-kinase activities have been studied using liposome substrates. This method, however, is hampered significantly by the labor-intensive manual lipid extraction followed by a low-throughput thin-layer chromatography analysis. The authors describe a high-throughput liposome substrate-based assay based on an automated lipid extraction method that allows them to study PI 3-kinase enzyme mechanism and quantitatively measure inhibitor activity using liposome substrates in a high-throughput mode. This improved assay format can easily be extended to study other classes of phosphoinositide lipid kinases.     

Insulin activates the alpha isoform of class II phosphoinositide 3-kinase.

The novel class II phosphoinositide (PI) 3-kinases are characterized by the presence of a C-terminal C2 domain, but little is known about their regulation. We find insulin causes a rapid 2-3-fold increase in the activity of PI 3-kinase C2alpha (PI3K-C2alpha) in CHO-IR cells, 3T3-L1 adipocytes, and fully differentiated L5L6 myotubes. No insulin-induced activation of PI3K-C2alpha was observed in cell types known to have low responsiveness to insulin including HEK 293 cells, 3T3-L1 preadipocytes, and undifferentiated L5L6 myoblasts. The mechanism of activation of PI3K-C2alpha by insulin differs from that of class Ia PI 3-kinases in that insulin stimulation did not cause PI3K-C2alpha to associate with IRS-1 or insulin receptor. PI3K-C2alpha existed as a doublet, and insulin stimulation caused a redistribution from the lower molecular weight band to the higher molecular weight band, suggesting phosphorylation-induced bandshift. Consistent with this, in vitro phosphatase treatment reduced the intensity of the upper band back to that seen in unstimulated cells. This suggests that insulin-induced phosphorylation could play a role in regulation of the activity of PI3K-C2alpha. The finding that insulin activates PI3K-C2alpha in cell types known to possess a wide range of responses to insulin suggests that PI3K-C2alpha is a novel component of insulin-stimulated signaling cascades.     

Specific requirement for the p85-p110alpha phosphatidylinositol 3-kinase during epidermal growth factor-stimulated actin nucleation in breast cancer cells

We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells with epidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nM wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110alpha and p110beta, and do not contain p110delta. Injection of specific inhibitory antibodies to p110alpha induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110beta antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110alpha antibodies. In summary, the p110alpha isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.     

TNF-alpha and leptin activate the alpha-isoform of class II phosphoinositide 3-kinase

The class II PI 3-kinases are known to be activated by growth factors and chemokines but to date there are no reports of cytokine mediated regulation. Further, the intracellular signalling mechanisms regulating the class-II PI 3-kinases are poorly understood. We investigated the effects of the cytokines TNFalpha and leptin on the activity of the alpha isoform of the class II PI 3-kinase (PI3K-C2alpha) and find that these stimulate the enzyme 2-fold and 3-fold, in CHO cells and J774.2 macrophages, respectively. The stimulation by leptin was not accompanied by recruitment of any tyrosine phosphorylated proteins to PI3K-C2alpha and no shift in electrophoretic mobility was noted. Furthermore, we demonstrate that the actions of both cytokines are blocked by the MEK inhibitor PD98059. These findings indicate that the cytokines activate PI3K-C2alpha and do so by a mechanism that requires activation of the ERK pathway and thus differs from the mechanism used by insulin to activate the enzyme.     

Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma). In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.     

Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation [1]. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Here, we report that primary murine bone marrow-derived macrophages (using alpha(v)beta(3) integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton [2, 3]. We show that inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process.     

A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.     

Optimal chemotactic responses of leukemic T cells to stromal cell-derived factor-1 requires the activation of both class IA and IB phosphoinositide 3-kinases

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are a multifunctional chemokine/receptor system with essential roles in the development of the immune system and other aspects of embryogenesis, including vascularization and organ development. SDF-1 is also a potent chemoattractant for T cells and has roles in both inflammation and immune homeostasis. Our group has previously demonstrated that phosphoinositide 3-kinase (PI 3-kinase) is activated in SDF-1-stimulated T cells and is indeed required for SDF-1-mediated chemotaxis. In this study Jurkat clones were established, stably expressing dominant negative constructs of class IA and class IB PI 3-kinases under the control of the tetracycline off inducible gene system, to determine the relative roles of these PI 3-kinases in SDF-1 signaling. Our results show that expression of either kinase-dead PI3Kgamma (KD-PI3Kgamma) or Deltap85 (a construct unable to bind class I(A) p110alpha, -beta, or -delta) leads to a partial inhibition of SDF-1-stimulated protein kinase B phosphorylation, but had no effect on SDF-1-induced phosphorylation of the mitogen-activated protein kinase ERK1/2. Functional studies demonstrated that expression of KD-PI3Kgamma markedly inhibited SDF-1-mediated chemotaxis, typically eliciting 40-60% inhibition. Interestingly, the expression of Deltap85 also leads to inhibition of the SDF-1-mediated chemotactic response, albeit to a much lesser extent than achieved with the KD-PI3Kgamma mutant, typically in the range of 20-40% inhibition. Furthermore, the inhibition of chemotaxis by the expression of dominant negative class IA or class IB PI 3-kinases could be enhanced by the presence of the PI 3-kinase inhibitor LY294002. Together, these results demonstrate that optimal chemotactic response of leukemic T cells to SDF-1 requires the activation of both class IA and class IB PI 3-kinases.