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.     

Class I phosphoinositide 3-kinases

Phosphoinositide 3-kinases (PI3-kinases) are a family of enzymes that 3'-phosphorylate the inositol head group of membrane phosphoinositides. They are subdivided into three major classes (I, II and III) based on their structural homology, regulation and substrate specificity. It is now becoming clear that PI3-kinase isoforms are subject to differential regulation and may play distinct roles within the cell. PI3-kinases and their second messenger lipid products have been implicated in a plethora of cellular responses with increasing evidence for involvement in the pathogenesis of human diseases. The future development of specific PI3-kinase isoform inhibitors may offer therapeutic benefit in a broad range of clinical settings, related to cancer, inflammatory and immunological diseases.     

Identification and characterization of a phosphoinositide phosphate kinase homolog

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) plays a central role in regulating the actin cytoskeleton as a substrate for phosphoinositide 3-kinase and phospholipase C as well as by binding directly to proteins that control the processes of actin monomer sequestration, filament severing, capping, nucleation, cross-linking, and bundling (Ma, L., Cantley, L. C., Janmey, P. A., and Kirschner, M. W. (1998) J. Cell Biol. 140, 1125-1136; Hinchliffe, K. (2000) Curr. Biol. 10, R104-R1051). Three related phosphatidylinositol 4-phosphate 5-kinases (PI(4)P 5-kinases) have been identified in mammalian cells (types Ialpha, Ibeta, and Igamma) and appear to play distinct roles in actin remodeling. Here we have identified a fourth member of this family by searching the human genome and EST data bases. This new protein, which we have designated phosphatidylinositol phosphate kinase homolog (PIPKH), is expressed at relatively high levels in brain and testis. Immunoprecipitates of PIPKH expressed in mammalian cells contain PI(4)P 5-kinase activity, but this activity is not affected by mutations in residues that inactivate other type I PI(4)P 5-kinases. We show that the PI(4)P 5-kinase activity in PIPKH immunoprecipitates can be explained by the ability of PIPKH to heterodimerize with other type I PI(4)P 5-kinases. Transfection of 293t cells with PIPKH resulted in >8-fold increase in total phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) without a significant net increase in total PI(4,5)P(2). When coexpressed with PIPKH, green fluorescent protein (GFP) fusion construct of the pleckstrin homology domain from Bruton's tyrosine kinase (GFP-BTK-PH) localized in intracellular vesicular structures, suggesting an unusual intracellular site of PI(3,4,5)P(3) production. Finally, expression of PIPKH induced the reorganization of actin from predominantly stress fibers to predominantly foci and comets similar to those observed previously in cells infected with the intracellular pathogen Listeria or transfected with recombinant PIPKIalpha. These results suggest that PIPKH acts as a scaffold to localize and regulate type I PI(4)P 5-kinases and the synthesis of PI(3,4,5)P(3).     

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.     

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.     

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.     

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.     

Identification of wortmannin-sensitive targets in 3T3-L1 adipocytes. DissociationoOf insulin-stimulated glucose uptake and glut4 translocation.

The current studies investigated the contribution of phosphatidylinositol 3-kinase (PI3-kinase) isoforms to insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation. Experiments involving the microinjection of antibodies specific for the p110 catalytic subunit of class I PI3-kinases demonstrated an absolute requirement for this form of the enzyme in GLUT4 translocation. This finding was confirmed by the demonstration that the PI3-kinase antagonist wortmannin inhibits GLUT4 and insulin-responsive aminopeptidase translocation with a dose response identical to that required to inhibit another class I PI3-kinase-dependent event, activation of pp70 S6-kinase. Interestingly, wortmannin inhibited insulin-stimulated glucose uptake at much lower doses, suggesting the existence of a second, higher affinity target of the drug. Subsequent removal of wortmannin from the media shifted this dose-response curve to one resembling that for GLUT4 translocation and pp70 S6-kinase. This is consistent with the lower affinity target being p110, which is irreversibly inhibited by wortmannin. Wortmannin did not reduce glucose uptake in cells stably expressing Myr-Akt, which constitutively induced GLUT4 translocation to the plasma membrane; this demonstrates that wortmannin does not inhibit the transporters directly. In addition to elucidating a second wortmannin-sensitive pathway in 3T3-L1 adipocytes, these studies suggest that the presence of GLUT4 on the plasma membrane is not sufficient for activation of glucose uptake.     

Crystal structure of the C2 domain of class II phosphatidylinositide 3-kinase C2alpha

Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3'-position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the C terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK-C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK-C2 domain has a typical anti-parallel beta-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfate-binding sites. On the basis of the structural features of these sulfate-binding sites, we have studied the lipid binding properties of the mCPK-C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to PtdIns(3,4)P(2) and PtdIns(4,5)P(2) and that three lysine residues at SBS I site, Lys-1420, Lys-1432, and Lys-1434, are responsible for the phospholipid binding affinity.     

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.     

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.     

Association of phosphatidylinositol 3-kinase composed of p110beta-catalytic and p85-regulatory subunits with the small GTPase Rab5.

A family of phosphatidylinositol 3-kinases (PI 3-kinase), comprising three major classes (I-III) in terms of substrate specificity and regulation, play important roles in a variety of cell functions. We previously reported that the class-I heterodimeric PI 3-kinase consisting of p110beta-catalytic and p85-regulatory subunits is synergistically activated by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating trimeric G proteins. Here we report an additional unique feature of the p110beta/p85 PI 3-kinase. The small GTPase Rab5 was identified as a binding protein for the p110beta-catalytic subunit in a yeast two-hybrid screening system. The interaction appears to require at least two separated amino-acid sequences present specifically in the beta isoform of p110 and the GTP-bound form of Rab5. The expressions of constitutively active and dominant negative mutants of Rab5 in THP-1 cells induce the stimulation and inhibition, respectively, of protein kinase B activity, which is dependent on the PI 3-kinase product phosphatidylinositol 3,4,5-triphosphate. These results suggest that there is a specific interaction between GTP-bound Rab5 and the p110beta/p85 PI 3-kinase, leading to efficient coupling of the lipid kinase product to its downstream target, protein kinase B.     

Differential activation of the PI 3-kinase effectors AKT/PKB and p70 S6 kinase by compound 48/80 is mediated by PKCalpha

The secretagogue compound 48/80 (c48/80) is a well known activator of calcium mediated processes and PKCs, and is a potent inducer of mast cell degranulation. As the latter process is a phosphoinositide 3-kinase (PI 3-kinase) mediated event, we wished to address whether or not c48/80 was an activator of PI 3-kinases. The data presented here reveal that c48/80 is an effective activator of PI 3-kinases as judged by the increased phosphorylation of PKB and p70(S6K) in fibroblasts in a PI 3-kinase dependent fashion. Compound 48/80 effectively translocates PKB to the plasma membrane and induces phosphorylation at serine 473 (S473), detected by fluorescence imaging of fixed cells. At higher concentrations the secretagogue is inhibitory towards PKB phosphorylation on S473. Conversely, p70(S6K) phosphorylation on T389 is unaffected at high doses. We provide evidence that the differential effect on the two PI 3-kinase effectors is due to activation of PKCalpha by c48/80, itself a PI 3-kinase dependent process. We conclude that compound 48/80 is an effective activator of PI 3-kinase dependent pathways, leading to the activation of effectors including PKB/Akt, p70(S6K) and PKCalpha. The latter is only activated by higher doses of c48/80 resulting in an inhibition of the c48/80 induced PKB phosphorylation, thus explaining the observed biphasic activation profile for PKB in response to this secretagogue.     

A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases

Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.     

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.     

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.     

Phosphatidylinositol 4-Kinases and PI4P Metabolism in the Nervous System: Roles in Psychiatric and Neurological Diseases

The four mammalian phosphatidylinositol 4-kinases, together with the PI(4,5)P₂ depleting 5-phosphatases of the oculocerebrorenal syndrome of Lowe and synaptojanin families, modulate neuronal pools of PI4P lipid and regulate intracellular membrane trafficking in the endocytic and secretory pathways. Dysfunctions in these enzymes have been associated with a broad spectrum of disorders including schizophrenia, bipolar disorder, Lowe syndrome, age-related neurodegeneration, Alzheimer’s disease and Down syndrome. Recent work has shown that reduced expression of individual phosphatidylinositol 4-kinase isozymes is associated with impaired survival of specific neuronal populations within the CNS. Furthermore, alterations to the concentrations of different phosphoinositide lipid species in the brain and, in particular, the ratio of PI4P to PI(4,5)P₂ can have deleterious effects on clathrin-dependent membrane trafficking both in the Golgi–endosomal pathway and at the plasma membrane. In this article, we focus on the cell biology, biochemistry and neuronal functions of the phosphatidylinositol 4-kinases and their emerging roles in psychiatric and neurological pathologies.     

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.