Background: PI4KA is a critical host factor for replication of hepatitis C virus in liver and a potential therapeutic target. Results: PI4KA inhibitors prevent the maintenance of PtdIns(4,5)P 2 pools during strong PLC activation. Conclusion: PI4KA plays a critical role in maintaining plasma membrane phosphoinositide pools. Significance: Safe pharmacological targeting of PI4KA is not feasible.

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 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 homogenous 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 similarly to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of > 0.7 for PI4KA in 384-well format, demonstrating its suitability for high-throughput screening applications.

Rationale AMP-activated protein kinase (AMPK) 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 upon AMPK activation. Nuclear accumulation of AMPK complexes containing γ2-subunit phosphorylated and inactivated RNA Pol I-associated transcription factor TIF-IA at Ser-635, precluding the assembly of transcription initiation complexes for rDNA. The subsequent down-regulation of pre-rRNA level led to attenuated 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 (cKO). Although the total AMPK activity was unaltered in cKO hearts due to upregulation of γ1-AMPK, the lack of γ2-AMPK sensitizes the heart to myocardial ischemia/reperfusion (I/R) injury. The cKO failed to suppress pre-rRNA level during I/R, and showed a greater infarct size. Conversely, cardiac-specific overexpression of γ2-AMPK decreased ribosome biosynthesis and ER stress during I/R 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 I/R injury. Our study reveals an isoform-specific function of γ2-AMPK in modulating ribosome biosynthesis, cell survival and cardioprotection.

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-proteincoupled receptors in higher eukaryotes.

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, ~ 230 kDa (isoform 2) and a shorter splice variant containing only the ~97 kDa C-terminus that includes the catalytic domain (isoform 1). However, Northern analysis of human tissues and cancer cells showed only a single transcript of ~ 7.5 kb with the exception of the proerythroleukemia line K562, which contained significantly higher level of the 7.5 kb transcript along with smaller ones of 2.4, 3.5 and 4.2 kb 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 ~97 kDa 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.

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Type II phosphatidylinositol 4-kinase β (PtdIns 4-kinase II β) is an enigma among the phosphatidylinositol 4-kinase family. The role of PtdIns 4-kinase II β in MCF-7 cells was addressed with the help of short hairpin RNA (shRNA). PtdIns 4-kinase II β 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 β shRNA-induced apoptosis. Caspase 8 and 9 inhibitors rescued MCF-7 cells from apoptosis. Shotgun proteomic studies with Flag-tagged PtdIns 4-kinase II β 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 β from MCF-7 cells. Deletion of membrane localization motif (ΔCCPCC) or a mutation in ATP-binding region (D304A) of PtdIns 4-kinase II β did not affect its interaction with Par-4. Pull-down assays with GST-PtdIns 4-kinase II β-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 β 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 β shRNA-induced apoptosis. These results suggest that PtdIns 4-kinase II β may be a novel regulator of Par-4 through protein–protein interactions. These studies have potential implications in cancer therapy.

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