Silencing of type II phosphatidylinositol 4-kinase ß stabilizes prostate apoptosis response-4 and induces apoptosis in cancer cells.

Type II phosphatidylinositol 4-kinase ß (PtdIns 4-kinase II ß) is an enigma among the phosphatidylinositol 4-kinase family. The role of PtdIns 4-kinase II ß in MCF-7 cells was addressed with the help of short hairpin RNA (shRNA). PtdIns 4-kinase II ß shRNA transfection increased pan-caspase activity and induced apoptosis in cancerous MCF-7 cells. Non-cancerous MCF-10A cells were resistant to PtdIns 4-kinase II ß shRNA-induced apoptosis. Caspase 8 and 9 inhibitors rescued MCF-7 cells from apoptosis. Shotgun proteomic studies with Flag-tagged PtdIns 4-kinase II ß immunoprecipitates showed tumor suppressor prostate apoptosis response-4 (Par-4) as one of the interacting proteins in HEK293 cells. In reciprocal experiments, Par-4 antibodies co-precipitated PtdIns 4-kinase II ß from MCF-7 cells. Deletion of membrane localization motif (ΔCCPCC) or a mutation in ATP-binding region (D304A) of PtdIns 4-kinase II ß did not affect its interaction with Par-4. Pull-down assays with GST-PtdIns 4-kinase II ß-truncated mutants showed that the region between 101 and 215 amino acid residues is essential for interaction with Par-4. At molecular level, PtdIns 4-kinase II ß shRNA transfection increased Par-4 stability, its nuclear localization and inhibition of NF-κB binding to target DNA. Knocking down of Par-4 with siRNA (small interfering RNA) rescued MCF-7 cells from PtdIns 4-kinase II ß shRNA-induced apoptosis. These results suggest that PtdIns 4-kinase II ß may be a novel regulator of Par-4 through protein-protein interactions. These studies have potential implications in cancer therapy.

Activation of γ2-AMPK Suppresses Ribosome Biogenesis and Protects Against Myocardial Ischemia/Reperfusion Injury.

RATIONALE: AMPK (AMP-activated protein kinase) is a heterotrimeric protein that plays an important role in energy homeostasis and cardioprotection. Two isoforms of each subunit are expressed in the heart, but the isoform-specific function of AMPK remains unclear. OBJECTIVE: We sought to determine the role of γ2-AMPK in cardiac stress response using bioengineered cell lines and mouse models containing either isoform of the γ-subunit in the heart. METHODS AND RESULTS: We found that γ2 but not γ1 or γ3 subunit translocated into nucleus on AMPK activation. Nuclear accumulation of AMPK complexes containing γ2-subunit phosphorylated and inactivated RNA Pol I (polymerase I)-associated transcription factor TIF-IA at Ser-635, precluding the assembly of transcription initiation complexes for rDNA. The subsequent downregulation of pre-rRNA level led to attenuated endoplasmic reticulum (ER) stress and cell death. Deleting γ2-AMPK led to increases in pre-rRNA level, ER stress markers, and cell death during glucose deprivation, which could be rescued by inhibition of rRNA processing or ER stress. To study the function of γ2-AMPK in the heart, we generated a mouse model with cardiac-specific deletion of γ2-AMPK (cardiac knockout [cKO]). Although the total AMPK activity was unaltered in cKO hearts because of upregulation of γ1-AMPK, the lack of γ2-AMPK sensitizes the heart to myocardial ischemia/reperfusion injury. The cKO failed to suppress pre-rRNA level during ischemia/reperfusion and showed a greater infarct size. Conversely, cardiac-specific overexpression of γ2-AMPK decreased ribosome biosynthesis and ER stress during ischemia/reperfusion insult, and the infarct size was reduced. CONCLUSIONS: The γ2-AMPK translocates into the nucleus to suppress pre-rRNA transcription and ribosome biosynthesis during stress, thus ameliorating ER stress and cell death. Increased γ2-AMPK activity is required to protect against ischemia/reperfusion injury. Our study reveals an isoform-specific function of γ2-AMPK in modulating ribosome biosynthesis, cell survival, and cardioprotection.

EFR3s are palmitoylated plasma membrane proteins that control responsiveness to G-protein-coupled receptors.

The yeast Efr3p protein is a main regulator of the Stt4p phosphatidylinositol 4-kinase at contact sites between the endoplasmic reticulum and the plasma membrane. A mutation in its fly homologue Rbo, leads to diminished light responses in the eye attributed to progressively impaired PLC signaling. Here, we find that Efr3s plays a role in maintaining responsiveness to the type-I angiotensin II (AngII) receptors. siRNA-mediated depletion of EFR3A and EFR3B impaired the sustained phase of cytosolic Ca(2+) response to high concentration of AngII in HEK293 cells that express wild type but not truncated AGTR1 (AT1a receptor), missing the phosphorylation sites. Efr3 depletion had minimal effect on the recovery of plasma membrane phosphoinositides during stimulation, and AT1 receptors still underwent ligand-induced internalization. A higher level of basal receptor phosphorylation and a larger response was observed after stimulation. Moreover, Gq activation more rapidly desensitized after AngII stimulation in Efr3 downregulated cells. A similar but less pronounced effect of EFR3 depletion was observed on the desensitization of the cAMP response after stimulation with isoproterenol. These data suggest that mammalian Efr3s contribute to the control of the phosphorylation state and, hence, desensitization of AT1a receptors, and could affect responsiveness of G-protein-coupled receptors in higher eukaryotes.

Type II PtdIns 4-kinase ß associates with CD4-p56lck complex and is involved in CD4 receptor signaling.

Type II phosphatidylinositol (PtdIns) 4-kinases are involved in the synthesis of PtdIns 4-phosphates and modulate various cell functions like, intracellular signaling, cytoskeletal rearrangements, vesicular trafficking, and pathogen invasion. In CD3 receptor activated T cells, a type II PtdIns 4-kinase ß is recruited to CD3 receptor zeta and plays a role in intracellular calcium release and probably in actin cytoskeleton reorganization. T cell receptor mediated activation is supported by CD4 receptor. The role of type II PtdIns 4-kinase ß in CD4 receptor-mediated signaling was addressed in the present manuscript. Crosslinking of CD4 receptors with monoclonal antibodies showed an increase in CD4-associated PtdIns 4-kinase activity and requires p56(lck) activity. Biochemical characterization suggests that it belongs to type II PtdIns 4-kinase family. shRNA mediated knockdown of type II PtdIns 4-kinase ß showed abrogation of CD4 receptor induced intracellular calcium release. These results suggest that type II PtdIns 4-kinase ß plays an integral part in CD4 receptor-mediated signaling.

Piperine inhibits type II phosphatidylinositol 4-kinases: a key component in phosphoinositides turnover.

Piperine has been shown to have anti-inflammatory properties. The molecular mechanisms by which it mediates anti-inflammatory activities remain elusive. Type II phosphatidylinositol 4-kinase(s) are key components in FcεRI receptor-mediated signaling leading to inflammatory mediators release in RBL-2H3 cells. The effects of piperine on IgE-mediated signaling and mast cell degranulation were investigated. Pretreatment of RBL-2H3 cells with piperine inhibited IgE-induced activation of type II PtdIns 4-kinase(s). In vitro lipid kinase assays showed piperine-inhibited type II PtdIns 4-kinase activity in a dose-dependent fashion with no effect on PtdIns 3-kinase activity. Concomitantly, pretreatment of RBL-2H3 cells with piperine also inhibited IgE-induced ß-hexosaminidase release in RBL-2H3 cells. These results suggest that type II PtdIns 4-kinases are part of piperine-mediated anti-inflammatory signaling mechanisms.

Type II phosphatidylinositol 4-kinases interact with FcεRIγ subunit in RBL-2H3 cells.

Ligation of high-affinity IgE receptor I (FcεRI) on RBL-2H3 cells leads to recruitment of FcεRI and type II phosphatidylinositol 4-kinases (PtdIns 4-kinases) into lipid rafts. Lipid raft integrity is required for the activation of type II PtdIns 4-kinases and signal transduction through FcεRIγ during RBL-2H3 cell activation. However, the molecular mechanism by which PtdIns 4-kinases are coupled to FcεRI signaling is elusive. Here, we report association of type II PtdIns 4-kinase activity with FcεRIγ subunit in anti-FcεRIγ immunoprecipitates. FcεRIγ-associated PtdIns 4-kinase activity increases threefold upon FcεRI ligation in anti-FcεRIγ immunoprecipitates. Biochemical characterization of PtdIns 4-kinase activity associated with FcεRIγ reveals that it is a type II PtdIns 4-kinases. Canonical tyrosine residues mutation in FcεRIγ ITAM (Y65 and Y76) reveals that these two tyrosine residues in γ subunit are required for its interaction with type II PtdIns 4-kinases.

Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate levels.

Phosphatidylinositol 4-kinase type IIIα (PI4KA) is a host factor essential for hepatitis C virus replication and hence is a target for drug development. PI4KA has also been linked to endoplasmic reticulum exit sites and generation of plasma membrane phosphoinositides. Here, we developed highly specific and potent inhibitors of PI4KA and conditional knock-out mice to study the importance of this enzyme in vitro and in vivo. Our studies showed that PI4KA is essential for the maintenance of plasma membrane phosphatidylinositol 4,5-bisphosphate pools but only during strong stimulation of receptors coupled to phospholipase C activation. Pharmacological blockade of PI4KA in adult animals leads to sudden death closely correlating with the drug's ability to induce phosphatidylinositol 4,5-bisphosphate depletion after agonist stimulation. Genetic inactivation of PI4KA also leads to death; however, the cause in this case is due to severe intestinal necrosis. These studies highlight the risks of targeting PI4KA as an anti-hepatitis C virus strategy and also point to important distinctions between genetic and pharmacological studies when selecting host factors as putative therapeutic targets.

Fyn kinase regulates type II PtdIns 4-kinases in RBL 2H3 cells.

Type II phosphatidylinositol 4-kinases are implicated in FcεRI-mediated signaling cascades leading to release of inflammatory molecules. Cross-linking of FcεRI on RBL 2H3 cells results in protein tyrosine phosphorylation and activation of type II PtdIns 4-kinase activity. Protein tyrosine kinase(s) that phosphorylate type II PtdIns 4-kinase(s) in RBL 2H3 cells remains elusive and is being addressed in this manuscript. Anti-Fyn kinase antibodies co-immunoprecipitated type II PtdIns 4-kinase activity from FcεRI cross-linked RBL 2H3 cells. In reciprocal assays, His-tagged types II PtdIns 4-kinases were shown to pull down Fyn kinase. Further, anti-Fyn immunoprecipitates were shown to phosphorylate type II PtdIns 4-kinase α and ß in in vitro assays. Pull down studies with GST-Fyn-SH2 and GST-Fyn-SH3 domains showed that type II PtdIns 4-kinases associate with Fyn-SH2 domain. Knockdown of Fyn kinase in RBL 2H3 cells abrogated activation of type II PtdIns 4-kinase activity in response to FcεRI cross-linking and type II PtdIns 4-kinase activity in anti-phosphotyrosine immunoprecipitates. Knockdown of Fyn kinase was also strongly correlated with a reduction in ß-hexosaminidase release in response to FcεRI cross-linking. These results suggest that type II PtdIns 4-kinases act downstream of Fyn kinase in FcεRI signaling cascades and are regulated by Fyn kinase.

Sanguinarine suppresses IgE induced inflammatory responses through inhibition of type II PtdIns 4-kinase(s).

The effects of sanguinarine on IgE mediated early signaling mechanisms leading to inflammatory mediators release were investigated. Pretreatment of RBL 2H3 cells with sanguinarine inhibited IgE induced activation of type II PtdIns 4-kinase activity. Concomitant with type II PtdIns 4-kinase inhibition, sanguinarine also inhibited IgE induced degranulation and ß hexosaminidase release in RBL 2H3 cells. In vitro assays showed sanguinarine inhibited type II PtdIns 4-kinase activity in a dose dependent fashion with no effect on PtdIns 3-kinase activity. Fluorescence spectroscopic studies suggested that sanguinarine binds to type II PtdIns 4-kinases α and ß isoforms with a Kd of 2.4 and 1.8µM, respectively. Kinetic studies showed that sanguinarine competes with PtdIns binding site of type II PtdIns 4-kinase ß. These results suggest that the anti-inflammatory effects of sanguinarine on PtdIns 3-kinase signaling pathway are more likely an indirect effect and emphasize the importance of the cross talk between type II PtdIns 4-kinases and PtdIns 3-kinases.

Type II phosphatidylinositol 4-kinase ß is an integral signaling component of early T cell activation mechanisms.

The early signaling events in T cell activation through CD3 receptor include a rapid change in intra cellular free calcium concentration and reorganization of actin cytoskeleton. Phosphatidylinositol 4-kinases (PtdIns 4-kinases) are implicated as key components in these early signaling events. The role of type II PtdIns 4-kinase ß in CD3 receptor signaling was investigated with the help of short hairpin RNA sequences. Cross-linking of CD3 receptors on Jurkat T Cells with monoclonal antibodies showed an early increase in type II PtdIns 4-kinase activity and co-localization of type II PtdIns 4-kinase ß with CD3 ζ. Transfection of Jurkat T Cells with shRNAs inhibited CD3 receptor mediated type II PtdIns 4-kinase activation with a concomitant reduction in intra cellular calcium release, suggesting a role for type II PtdIns 4-kinase ß in CD3 receptor signal transduction. Knock-down of type II PtdIns 4-kinase ß with shRNAs also correlated with a decrease in PtdIns 4-kinase activity in cytoskeleton fractions and reduced adhesion to matrigel surfaces. These results indicate that type II PtdIns 4-kinase ß is a key component in early T cell activation signaling cascades.

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.

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.

Genetic and functional studies of phosphatidyl-inositol 4-kinase type IIIα.

Phosphatidylinositol 4-kinase type IIIa (PI4KIIIα) is one of four mammalian PI 4-kinases that catalyzes the first committed step in polyphosphoinositide synthesis. PI4KIIIα has been linked to regulation of ER exit sites and to the synthesis of plasma membrane phosphoinositides and recent studies have also revealed its importance in replication of the Hepatitis C virus in liver. Two isoforms of the mammalian PI4KIIIα have been described and annotated in GenBank: a larger, ~230kDa (isoform 2) and a shorter splice variant containing only the ~97kDa C-terminus that includes the catalytic domain (isoform 1). However, Northern analysis of human tissues and cancer cells showed only a single transcript of ~7.5kb with the exception of the proerythroleukemia line K562, which contained significantly higher level of the 7.5kb transcript along with smaller ones of 2.4, 3.5 and 4.2kb size. Bioinformatic analysis also confirmed the high copy number of PI4KIIIα transcript in K562 cells along with several genes located in the same region in Chr22, including two pseudogenes that cover most exons coding for isoform 1, consistent with chromosome amplification. A panel of polyclonal antibodies raised against peptides within the C-terminal half of PI4KIIIα failed to detect the shorter isoform 1 either in COS-7 cells or K562 cells. Moreover, expression of a cDNA encoding isoform 1 yielded a protein of ~97kDa that showed no catalytic activity and failed to rescue hepatitis C virus replication. These data draw attention to PI4KIIIα as one of the genes found in Chr22q11, a region affected by chromosomal instability, but do not substantiate the existence of a functionally relevant short form of PI4KIIIα.