Structure-based design and modular synthesis of novel PI4K class II inhibitors bearing a 4-aminoquinazoline scaffold.

Novel 4-aminoquinazoline-6-carboxamide derivatives bearing differently substituted aryl or heteroaryl groups at position 7 in the core were rationally designed, synthesized and evaluated for biological activity in vitro as phosphatidylinositol 4-kinase IIα (PI4K2A) inhibitors. The straightforward approach described here enabled the sequential, modular synthesis and broad functionalization of the scaffold in a mere six steps. The SAR investigation reported here is based on detailed structural analysis of the conserved binding mode of ATP and other adenine derivatives to the catalytic site of type II PI4Ks, combined with extensive docking studies. Several compounds exhibited significant activity against PI4K2A. Moreover, we solved a crystal structure of PI4K2B in complex with one of our lead ligand candidates, which validated the ligand binding site and pose predicted by our docking-based ligand model. These discoveries suggest that our structure-based approach may be further developed and employed to synthesize new inhibitors with optimized potency and selectivity for this class of PI4Ks.

The chemistry and biology of phosphatidylinositol 4-phosphate at the plasma membrane.

Phosphoinositides are an important class of anionic, low abundance signaling lipids distributed throughout intracellular membranes. The plasma membrane contains three phosphoinositides: PI(4)P, PI(4,5)P2, and PI(3,4,5)P3. Of these, PI(4)P has remained the most mysterious, despite its characterization in this membrane more than a half-century ago. Fortunately, recent methodological innovations at the chemistry-biology interface have spurred a renaissance of interest in PI(4)P. Here, we describe these new toolsets and how they have revealed novel functions for the plasma membrane PI(4)P pool. We examine high-resolution structural characterization of the plasma membrane PI 4-kinase complex that produces PI(4)P, tools for modulating PI(4)P levels including isoform-selective PI 4-kinase inhibitors, and fluorescent probes for visualizing PI(4)P. Collectively, these chemical and biochemical approaches have revealed insights into how cells regulate synthesis of PI(4)P and its downstream metabolites as well as new roles for plasma membrane PI(4)P in non-vesicular lipid transport, membrane homeostasis and trafficking, and cell signaling pathways.

Architecture of the human PI4KIIIα lipid kinase complex.

Plasma membrane (PM) phosphoinositides play essential roles in cell physiology, serving as both markers of membrane identity and signaling molecules central to the cell's interaction with its environment. The first step in PM phosphoinositide synthesis is the conversion of phosphatidylinositol (PI) to PI4P, the precursor of PI(4,5)P2 and PI(3,4,5)P3 This conversion is catalyzed by the PI4KIIIα complex, comprising a lipid kinase, PI4KIIIα, and two regulatory subunits, TTC7 and FAM126. We here report the structure of this complex at 3.6-Å resolution, determined by cryo-electron microscopy. The proteins form an obligate ∼700-kDa superassembly with a broad surface suitable for membrane interaction, toward which the kinase active sites are oriented. The structural complexity of the assembly highlights PI4P synthesis as a major regulatory junction in PM phosphoinositide homeostasis. Our studies provide a framework for further exploring the mechanisms underlying PM phosphoinositide regulation.

Multistep Compositional Remodeling of Supported Lipid Membranes by Interfacially Active Phosphatidylinositol Kinases.

The multienzyme catalytic phosphorylation of phosphatidylinositol (PI) in a supported lipid membrane platform is demonstrated for the first time. One-step treatment with PI 4-kinase IIIß (PI4Kß) yielded PI 4-phosphate (PI4P), while a multistep enzymatic cascade of PI4Kß followed by PIP 5-kinase produced PI-4,5-bisphosphate (PI(4,5)P2 or PIP2). By employing quartz crystal microbalance with dissipation monitoring, we were able to track membrane association of kinase enzymes for the first time as well as detect PI4P and PI(4,5)P2 generation based on subsequent antibody binding to the supported lipid bilayers. Pharmacologic inhibition of PI4Kß by a small molecule inhibitor was also quantitatively assessed, yielding an EC50 value that agrees well with conventional biochemical readout. Taken together, the development of a PI-containing supported membrane platform coupled with surface-sensitive measurement techniques for kinase studies opens the door to exploring the rich biochemistry and pharmacological targeting of membrane-associated phosphoinositides.

The high-resolution crystal structure of phosphatidylinositol 4-kinase IIß and the crystal structure of phosphatidylinositol 4-kinase IIα containing a nucleoside analogue provide a structural basis for isoform-specific inhibitor design.

Phosphatidylinositol 4-phosphate (PI4P) is the most abundant monophosphoinositide in eukaryotic cells. Humans have four phosphatidylinositol 4-kinases (PI4Ks) that synthesize PI4P, among which are PI4K IIß and PI4K IIα. In this study, two crystal structures are presented: the structure of human PI4K IIß and the structure of PI4K IIα containing a nucleoside analogue. The former, a complex with ATP, is the first high-resolution (1.9 Å) structure of a PI4K. These structures reveal new details such as high conformational heterogeneity of the lateral hydrophobic pocket of the C-lobe and together provide a structural basis for isoform-specific inhibitor design.

Targeting Plasmodium PI(4)K to eliminate malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Mammalian phosphatidylinositol 4-kinases as modulators of membrane trafficking and lipid signaling networks.

The four mammalian phosphatidylinositol 4-kinases modulate inter-organelle lipid trafficking, phosphoinositide signalling and intracellular vesicle trafficking. In addition to catalytic domains required for the synthesis of PI4P, the phosphatidylinositol 4-kinases also contain isoform-specific structural motifs that mediate interactions with proteins such as AP-3 and the E3 ubiquitin ligase Itch, and such structural differences determine isoform-specific roles in membrane trafficking. Moreover, different permutations of phosphatidylinositol 4-kinase isozymes may be required for a single cellular function such as occurs during distinct stages of GPCR signalling and in Golgi to lysosome trafficking. Phosphatidylinositol 4-kinases have recently been implicated in human disease. Emerging paradigms include increased phosphatidylinositol 4-kinase expression in some cancers, impaired functioning associated with neurological pathologies, the subversion of PI4P trafficking functions in bacterial infection and the activation of lipid kinase activity in viral disease. We discuss how the diverse and sometimes overlapping functions of the phosphatidylinositol 4-kinases present challenges for the design of isoform-specific inhibitors in a therapeutic context.

A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32)P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50) of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

Phosphatidylinositol 4-kinase IIα is palmitoylated by Golgi-localized palmitoyltransferases in cholesterol-dependent manner.

Phosphatidylinositol 4-kinase IIα (PI4KIIα) is predominantly Golgi-localized, and it generates >50% of the phosphatidylinositol 4-phosphate in the Golgi. The lipid kinase activity, Golgi localization, and "integral" membrane binding of PI4KIIα and its association with low buoyant density "raft" domains are critically dependent on palmitoylation of its cysteine-rich (173)CCPCC(177) motif and are also highly cholesterol-dependent. Here, we identified the palmitoyl acyltransferases (Asp-His-His-Cys (DHHC) PATs) that palmitoylate PI4KIIα and show for the first time that palmitoylation is cholesterol-dependent. DHHC3 and DHHC7 PATs, which robustly palmitoylated PI4KIIα and were colocalized with PI4KIIα in the trans-Golgi network (TGN), were characterized in detail. Overexpression of DHHC3 or DHHC7 increased PI4KIIα palmitoylation by >3-fold, whereas overexpression of the dominant-negative PATs or PAT silencing by RNA interference decreased PI4KIIα palmitoylation, "integral" membrane association, and Golgi localization. Wild-type and dominant-negative DHHC3 and DHHC7 co-immunoprecipitated with PI4KIIα, whereas non-candidate DHHC18 and DHHC23 did not. The PI4KIIα (173)CCPCC(177) palmitoylation motif is required for interaction because the palmitoylation-defective SSPSS mutant did not co-immunoprecipitate with DHHC3. Cholesterol depletion and repletion with methyl-ß-cyclodextrin reversibly altered PI4KIIα association with these DHHCs as well as PI4KIIα localization at the TGN and "integral" membrane association. Significantly, the Golgi phosphatidylinositol 4-phosphate level was altered in parallel with changes in PI4KIIα behavior. Our study uncovered a novel mechanism for the preferential recruitment and activation of PI4KIIα to the TGN by interaction with Golgi- and raft-localized DHHCs in a cholesterol-dependent manner.

Metabolism of phosphatidylinositol 4-kinase IIIα-dependent PI4P Is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity.

4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase α (PI4KIIIα) has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIα and, to a lesser extent, PI4KIIIß. In Huh7.5 cells, PI4KIIIα is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIIIα also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIIIß isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIIIα by RNA interference. In light of our findings, we propose that the antiviral effect of 4-anilino quinazoline compounds is mediated by the inhibition of PI4KIIIα and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV--by recruiting PI4KIIIα in the RNA replication complex--hijacks PI4P metabolism, ultimately resulting in a markedly altered subcellular distribution of the PI4KIIIα product.

The phosphatidylinositol 4-kinases: don't call it a comeback.

Phosphatidylinositol 4-phosphate (PtdIns4P) is a quantitatively minor membrane phospholipid which is the precursor of PtdIns(4,5)P (2) in the classical agonist-regulated phospholipase C signalling pathway. However, PtdIns4P also governs the recruitment and function of numerous trafficking molecules, principally in the Golgi complex. The majority of phosphoinositides (PIs) phosphorylated at the D4 position of the inositol headgroup are derived from PtdIns4P and play roles in a diverse array of fundamental cellular processes including secretion, cell migration, apoptosis and mitogenesis; therefore, PtdIns4P biosynthesis can be regarded as key point of regulation in many PI-dependent processes.Two structurally distinct sequence families, the type II and type III PtdIns 4-kinases, are responsible for PtdIns4P synthesis in eukaryotic organisms. These important proteins are differentially expressed, localised and regulated by distinct mechanisms, indicating that the enzymes perform non-redundant roles in trafficking and signalling. In recent years, major advances have been made in our understanding of PtdIns4K biology and here we summarise current knowledge of PtdIns4K structure, function and regulation.

Characterization of molecular recognition of phosphoinositide-3-kinase α inhibitor through molecular dynamics simulation.

Phosphatidylinositol 3-kinase α (PI3Kα) is a promising target for anticancer drug discovery due to its overactivation in tumor cells. To systematically investigate the interactions between PI3Kα and PIK75 which is the most selective PI3Kα inhibitor reported to date, molecular docking, molecular dynamics simulation, and ensuing energetic analysis were utilized. The binding free energy between PI3Kα and PIK75 is -10.04 kcal•mol(-1) using MMPBSA method, while -13.88 kcal•mol(-1) using MMGBSA method, which is beneficial for the binding. The van der Waals/hydrophobic and electrostatic interactions play critical roles for the binding. The binding mode of PIK75 for PI3Kα is predicted. The conserved hydrophobic adenine region of PI3Kα made up of Ile800, Ile848, Val850, Val851, Met922, Phe930, and Ile932 accommodates the flat 6-bromine imidazo[1,2-a]pyridine ring of PIK75. The 2-methyl-5-nitrophenyl group of PIK75 extends to the P-loop region, and has four hydrogen-bond arms with the backbone and side chain of Ser773 and Ser774. And the distinct conformation of the P-loop induced by PIK75 is speculated to be responsible for the selectivity profile of PIK75. The predicted binding mode of PIK75 for PI3Kα presented in this study may help design high affinity and selective compounds to target PI3Kα.

Epigallocatechin gallate (EGCG) inhibits type II phosphatidylinositol 4-kinases: a key component in pathways of phosphoinositide turnover.

Type II phosphatidylinositol (PtdIns) 4-kinases produce PtdIns 4-phosphate, an early key signaling molecule in phosphatidylinositol cycle, which is indispensable for T cell activation. Type II PtdIns 4-kinase alpha and beta have similar biochemical properties. To distinguish these isoforms Epigallocatechin gallate (EGCG) has been evaluated as a specific inhibitor. EGCG is the major active catechin in green tea having anti-inflammatory, antiatherogenic and cancer chemopreventive properties. The precise mechanism of actions and molecular targets of EGCG in early signaling cascades are not well understood. In the present study, we have shown that EGCG inhibits type II PtdIns 4-kinases (α and ß isoforms) and PtdIns 3-kinase activity in vitro. EGCG directly bind to both alpha and beta isoforms of type II PtdIns 4-kinases with a Kd of 2.62 µM and 1.02 µM, respectively. Type II PtdIns 4-kinase-EGCG complex have different binding pattern at its excited state. Both isoforms showed significant change in helicity upon binding with EGCG. EGCG modulates its effect by interacting with ATP binding pocket; the residues likely to be involved in EGCG binding were predicted by Autodock. Our findings suggest that EGCG inhibits two isoforms and could be a key to regulate T cell activation.

Palmitoylation controls the catalytic activity and subcellular distribution of phosphatidylinositol 4-kinase II{alpha}.

Phosphatidylinositol 4-kinases play essential roles in cell signaling and membrane trafficking. They are divided into type II and III families, which have distinct structural and enzymatic properties and are essentially unrelated in sequence. Mammalian cells express two type II isoforms, phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) and IIbeta (PI4KIIbeta). Nearly all of PI4KIIalpha, and about half of PI4KIIbeta, associates integrally with membranes, requiring detergent for solubilization. This tight membrane association is because of palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domains of both type II isoforms. Deletion of this motif from PI4KIIalpha converts the kinase from an integral to a tightly bound peripheral membrane protein and abrogates its catalytic activity ( Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Sudhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708 ). Here we identify the first two cysteines in the CCPCC motif as the principal sites of palmitoylation under basal conditions, and we demonstrate the importance of the central proline for enzymatic activity, although not for membrane binding. We further show that palmitoylation is critical for targeting PI4KIIalpha to the trans-Golgi network and for enhancement of its association with low buoyant density membrane fractions, commonly termed lipid rafts. Replacement of the four cysteines in CCPCC with a hydrophobic residue, phenylalanine, substantially restores catalytic activity of PI4KIIalpha in vitro and in cells without restoring integral membrane binding. Although this FFPFF mutant displays a perinuclear distribution, it does not strongly co-localize with wild-type PI4KIIalpha and associates more weakly with lipid rafts.

p85beta phosphoinositide 3-kinase regulates CD28 coreceptor function.

CD28 is a receptor expressed on T cells that regulates their differentiation after antigen stimulation to long-term-survival memory T cells. CD28 enhances T-cell receptor signals and reduces expression of CBL ubiquitin ligases, which negatively control T-cell activation. In the absence of CD28 ligation during the primary stimulation, CBL levels remain high and T cells fail to mount an efficient secondary response. CD28 associates with p85alpha, one of the regulatory subunits of phosphoinositide-3-kinase (PI3K), but the relevance of this interaction is debated. We examined here the contribution of the other ubiquitous PI3K regulatory subunit, p85beta, in CD28 function. We describe that p85beta bound to CD28 and to CBL with greater affinity than p85alpha. Moreover, deletion of p85beta impaired CD28-induced intracellular events, including c-CBL and CBL-b down-regulation as well as PI3K pathway activation. This resulted in defective differentiation of activated T cells, which failed to exhibit an efficient secondary immune response. Considering that p85beta-deficient T cells fail in recall responses and that p85beta binds to and regulates CD28 signals, the presented observations suggest the involvement of p85beta in CD28-mediated activation and differentiation of antigen-stimulated T cells.

Identification of phosphatidylinositol 4-kinase type II beta as HLA class II-restricted target in graft versus leukemia reactivity.

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLIs). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Because HLA class II molecules are predominantly expressed on hematopoietic cells, mHag-specific CD4(+) T cells may selectively mediate graft versus leukemia (GvL) reactivity without graft versus host disease (GvHD). In this study, we used a recombinant bacteria cDNA library for the identification of the first autosomal HLA class II (HLA-DQB1*0603)-restricted mHag LB-PI4K2B-1S encoded by the broadly expressed phosphatidylinositol 4-kinase type II beta gene. A polyclonal CD4(+) T cell response against LB-PI4K2B-1S was demonstrated in a patient with relapsed chronic myeloid leukemia (CML) who responded to DLI after HLA-matched alloSCT. LB-PI4K2B-1S-specific CD4(+) T cells recognized and lysed the CD34(+) CML cells of the patient and other leukemic cells as well as high HLA-DQ-expressing normal hematopoietic cells. HLA-DQ expression on normal cells of nonhematopoietic origin was moderately up-regulated by IFN-gamma and not sufficient for T cell recognition. We hypothesize that LB-PI4K2B-1S-specific CD4(+) T cells contributed to the antitumor response by both directly eliminating malignant cells as effector cells and stimulating CD8(+) T cell immunity as helper cells.

The oxysterol binding protein Kes1p regulates Golgi apparatus phosphatidylinositol-4-phosphate function.

The Saccharomyces cerevisiae phosphatidylcholine/phosphatidylinositol transfer protein Sec14p is required for Golgi apparatus-derived vesicular transport through coordinate regulation of phospholipid metabolism. Sec14p is normally essential. The essential requirement for SEC14 can be bypassed by inactivation of (i) the CDP-choline pathway for phosphatidylcholine synthesis or (ii) KES1, which encodes an oxysterol binding protein. A unique screen was used to determine genome-wide genetic interactions for the essential gene SEC14 and to assess whether the two modes of "sec14 bypass" were similar or distinct. The results indicate that inactivation of the CDP-choline pathway allows cells with inactivated SEC14 to live through a mechanism distinct from that of inactivation of KES1. We go on to demonstrate an important biological function of Kes1p. Kes1p regulates Golgi apparatus-derived vesicular transport by inhibiting the function of Pik1p-generated Golgi apparatus phosphatidylinositol-4-phosphate (PI-4P). Kes1p affects both the availability and level of Golgi apparatus PI-4P. A set of potential PI-4P-responsive proteins that include the Rab GTPase Ypt31p and its GTP exchange factor are described.

Structural insights into activation of phosphatidylinositol 4-kinase (Pik1) by yeast frequenin (Frq1).

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca2+-bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca2+ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices (125-135 and 156-169) connected by a 20-residue loop. Side chains in the Pik1 N-terminal helix (Val-127, Ala-128, Val-131, Leu-132, and Leu-135) interact with solvent-exposed residues in the Frq1 C-terminal pocket (Leu-101, Trp-103, Val-125, Leu-138, Ile-152, and Leu-155); side chains in the Pik1 C-terminal helix (Ala-157, Ala-159, Leu-160, Val-161, Met-165, and Met-167) contact solvent-exposed residues in the Frq1 N-terminal pocket (Trp-30, Phe-34, Phe-48, Ile-51, Tyr-52, Phe-55, Phe-85, and Leu-89). This defined complex confirms that residues in Pik1 pinpointed as necessary for Frq1 binding by site-directed mutagenesis are indeed sufficient for binding. Removal of the Pik1 N-terminal region (residues 8-760) from its catalytic domain (residues 792-1066) abolishes lipid kinase activity, inconsistent with Frq1 binding simply relieving an autoinhibitory constraint. Deletion of the lipid kinase unique motif (residues 35-110) also eliminates Pik1 activity. In the complex, binding of Ca2+-bound Frq1 forces the Pik1 chain into a U-turn. Frq1 may activate Pik1 by facilitating membrane targeting via the exposed N-myristoyl group and by imposing a structural transition that promotes association of the lipid kinase unique motif with the kinase domain.

Nuclear PtdIns5P as a transducer of stress signaling: an in vivo role for PIP4Kbeta.

Inhibitor of growth protein-2 (ING2) is a nuclear adaptor protein that can regulate p53 and histone acetylation in response to cellular stress and contains a PHD (plant homeodomain) finger that can interact with phosphatidylinositol-5-phosphate (PtdIns5P). However, whether or how nuclear PtdIns5P levels are regulated in response to cellular stress or whether ING2 can sense these changes has not been demonstrated. We show that UV irradiation increases nuclear PtdIns5P levels via inhibition of the activity of the beta isoform of PtdIns5P 4-kinase (PIP4Kbeta), an enzyme that can phosphorylate and remove PtdIns5P. Inhibition of PIP4Kbeta activity occurs through the direct phosphorylation of PIP4Kbeta at Ser326 by the p38 stress-activated protein kinase. Finally, we show that changes in nuclear PtdIns5P are translated into changes in the association of ING2 with chromatin. Our data define a pathway connecting cellular stressors with changes in nuclear PtdIns5P levels and the regulation of PHD motif-containing proteins.

Phosphatidylinositol 4-kinases: old enzymes with emerging functions.

Phosphoinositides account for only a tiny fraction of cellular phospholipids but are extremely important in the regulation of the recruitment and activity of many signaling proteins in cellular membranes. Phosphatidylinositol (PtdIns) 4-kinases generate PtdIns 4-phosphate, the precursor of important regulatory phosphoinositides but also an emerging regulatory molecule in its own right. The four mammalian PtdIns 4-kinases regulate a diverse array of signaling events, as well as vesicular trafficking and lipid transport, but the mechanisms by which their lipid product PtdIns 4-phosphate controls these processes is only beginning to unfold.