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.     

A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis

The endgame of cytokinesis can follow one of two pathways depending on developmental context: resolution into separate cells or formation of a stable intercellular bridge. Here we show that the four wheel drive (fwd) gene of Drosophila melanogaster is required for intercellular bridge formation during cytokinesis in male meiosis. In fwd mutant males, contractile rings form and constrict in dividing spermatocytes, but cleavage furrows are unstable and daughter cells fuse together, producing multinucleate spermatids. fwd is shown to encode a phosphatidylinositol 4-kinase (PI 4-kinase), a member of a family of proteins that perform the first step in the synthesis of the key regulatory membrane phospholipid PIP2. Wild-type activity of the fwd PI 4-kinase is required for tyrosine phosphorylation in the cleavage furrow and for normal organization of actin filaments in the constricting contractile ring. Our results suggest a critical role for PI 4-kinases and phosphatidylinositol derivatives during the final stages of cytokinesis.     

Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe, interacts with a phosphatidylinositol 4-kinase

The proposed function of Cdc4p, an essential contractile ring protein in Schizosaccharomyces pombe, is that of a myosin essential light chain. However, five conditionally lethal cdc4 alleles exhibit complementation in diploids. Such interallelic complementation is not readily explained if the sole function of Cdc4p is that of a myosin essential light chain. Complementation of cdc4 alleles could occur only if different mutant forms can assemble into an active oligomeric complex or if Cdc4p has more than one essential function. To search for other proteins that may interact with Cdc4p, we performed a two-hybrid screen and identified two such candidates: one similar to Saccharomyces cerevisiae Vps27p and the other a putative phosphatidylinositol (PI) 4-kinase. Binding of Cdc4p to the latter and to myosin heavy chain (Myo2p) was confirmed by immunosorbent assays. Deletion studies demonstrated interaction between the Cdc4p C-terminal domain and the PI 4-kinase C-terminal domain. Furthermore, interaction was abolished by the Cdc4p C-terminal domain point mutation, Gly107 to Ser. This allele also causes failure of cytokinesis. Ectopic expression of the PI 4-kinase C-terminal domain caused cytokinesis defects that were most extreme in cells carrying the G107S allele. We suggest that Cdc4p plays multiple roles in cytokinesis and that interaction with a PI 4-kinase may be important for contractile ring assembly and/or function.     

A beta-tubulin mutation selectively uncouples nuclear division and cytokinesis in Tetrahymena thermophila

The ciliated protozoan Tetrahymena thermophila contains two distinct nuclei within a single cell-the mitotic micronucleus and the amitotic macronucleus. Although microtubules are required for proper division of both nuclei, macronuclear chromosomes lack centromeres and the role of microtubules in macronuclear division has not been established. Here we describe nuclear division defects in cells expressing a mutant beta-tubulin allele that confers hypersensitivity to the microtubule-stabilizing drug paclitaxel. Macronuclear division is profoundly affected by the btu1-1 (K350M) mutation, producing cells with widely variable DNA contents, including cells that lack macronuclei entirely. Protein expressed by the btu1-1 allele is dominant over wild-type protein expressed by the BTU2 locus. Normal macronuclear division is restored when the btu1-1 allele is inactivated by targeted disruption or expressed as a truncated protein. Immunofluorescence studies reveal elongated microtubular structures that surround macronuclei that fail to migrate to the cleavage furrows. In contrast, other cytoplasmic microtubule-dependent processes, such as cytokinesis, cortical patterning, and oral apparatus assembly, appear to be unaffected in the mutant. Micronuclear division is also perturbed in the K350M mutant, producing nuclei with elongated early-anaphase spindle configurations that persist well after the initiation of cytokinesis. The K350M mutation affects tubulin dynamics, as the macronuclear division defect is exacerbated by three treatments that promote microtubule polymerization: (i) elevated temperatures, (ii) sublethal concentrations of paclitaxel, and (iii) high concentrations of dimethyl sulfoxide. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with 3-methyladenine or wortmannin also induces amacronucleate cell formation in a btu1-1-dependent manner. Conversely, the myosin light chain kinase inhibitor ML-7 has no effect on nuclear division in the btu1-1 mutant strain. These findings provide new insights into microtubule dynamics and link the evolutionarily conserved PI 3-kinase signaling pathway to nuclear migration and/or division in Tetrahymena.     

Retracted: Downregulated microRNA-135a ameliorates rheumatoid arthritis by inactivation of the phosphatidylinositol 3-kinase/AKT signaling pathway via phosphatidylinositol 3-kinase regulatory subunit 2

Synovial fibroblasts (SFs) of rheumatoid arthritis (RA) are phenotypically aggressive, typically progressing into arthritic cartilage degradation. Throughout our study, we made explorations into the effects of microRNA-135a (miR-135a) on the SFs involved in RA by mediating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway via regulation of phosphatidylinositol 3-kinase regulatory subunit 2 (PIK3R2). The expression of PI3K was higher, the expression of PIK3R2 was lower, and AKT was phosphorylated in the RA synovial tissues, relative to the levels found in the normal synovial tissues. We predicted miR-135a to be a candidate miR targeting PIK3R2 using an online website, microRNA.org, which was verified with a dual-luciferase reporter gene assay. Subsequently, high miR-135a expression was observed in RA synovial tissues. To study the effect of the interaction between miR-135a and PIK3R2 in RA, the SFs isolated from RA samples were cultured and transfected with mimic, inhibitor, and small interfering RNA. The proliferation, invasion, and apoptosis of the SFs were detected after the transfection. The cells transfected with miR-135a inhibitor showed inhibited cell proliferation, migration, and invasion, while also displaying promoted cell apoptosis, G0/G1 cell ratio, and decreased S cell ratio, through upregulation of PIK3R2 and inactivation of the PI3K/AKT signaling pathway. These findings provided evidence that downregulation of miR-135a inhibits proliferation, migration, and invasion and promotes apoptosis of SFs in RA by upregulating the PIK3R2 coupled with inactivating the PI3K/AKT signaling pathway. The downregulation of miR-135a might be a potential target in the treatment of RA.     

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.     

Ablation of phosphoinositide-3-kinase class II alpha suppresses hepatoma cell proliferation

Cancer such as hepatocellular carcinoma (HCC) is characterized by complex perturbations in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K/AKT) pathways. Herein we investigated the role of PI3K catalytic isoforms, particularly class II isoforms in HCC proliferation. Among the siRNAs tested against the eight known catalytic PI3K isoforms, specific ablation of class II PI3K alpha (PIK3C2α) was the most effective in impairing cell growth and this was accompanied by concomitant decrease in PIK3C2α mRNA and protein levels. Colony formation ability of cells deficient for PIK3C2α was markedly reduced and growth arrest was associated with increased caspase 3 levels. A small but significant difference in gene dosage and expression levels was detected between tumor and non-tumor tissues in a cohort of 19 HCC patients. Taken together, these data suggest for the first time that in addition to class I PI3Ks in cancer, class II PIK3C2α can modulate HCC cell growth.     

A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic

The mammalian class III phosphatidylinositol 3-kinase (PI3K-III) complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Recent studies suggest the existence of distinct PI3K-III sub-complexes that can potentially confer functional specificity. While a substantial body of work has focused on the roles of individual PI3K-III subunits in autophagy, functional studies on their contribution to endocytic receptor downregulation and cytokinesis are limited. We therefore sought to elucidate the specific nature of the PI3K-III complexes involved in these two processes. High-content microscopy-based assays combined with siRNA-mediated depletion of individual subunits indicated that a specific sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates both receptor degradation and cytokinesis, whereas ATG14L, a PI3K-III subunit involved in autophagy, is not required. The unanticipated role of UVRAG and BIF-1 in cytokinesis was supported by a strong localisation of these proteins to the midbody. Importantly, while the tumour suppressive functions of Beclin 1, UVRAG and BIF-1 have previously been ascribed to their roles in autophagy, these results open the possibility that they may also contribute to tumour suppression via downregulation of mitogenic signalling by growth factor receptors or preclusion of aneuploidy by ensuring faithful completion of cell division.     

Oncogenic PIK3CA-driven mammary tumors frequently recur via PI3K pathway-dependent and PI3K pathway-independent mechanisms

PIK3CA gain-of-function mutations are a common oncogenic event in human malignancy, making phosphatidylinositol 3-kinase (PI3K) a target for cancer therapy. Despite the promise of targeted therapy, resistance often develops, leading to treatment failure. To elucidate mechanisms of resistance to PI3K-targeted therapy, we constructed a mouse model of breast cancer conditionally expressing human PIK3CA(H1047R). Notably, most PIK3CA(H1047R)-driven mammary tumors recurred after PIK3CA(H1047R) inactivation. Genomic analyses of recurrent tumors revealed multiple lesions, including focal amplification of Met or Myc (also known as c-Met and c-Myc, respectively). Whereas Met amplification led to tumor survival dependent on activation of endogenous PI3K, tumors with Myc amplification became independent of the PI3K pathway. Functional analyses showed that Myc contributed to oncogene independence and resistance to PI3K inhibition. Notably, PIK3CA mutations and c-MYC elevation co-occur in a substantial fraction of human breast tumors. Together, these data suggest that c-MYC elevation represents a potential mechanism by which tumors develop resistance to current PI3K-targeted therapies.     

Genetic and bioinformatic analyses of the expression and function of PI3K regulatory subunit PIK3R3 in an Asian patient gastric cancer library

ABSTRACT: BACKGROUND: While there is strong evidence for phosphatidylinositol 3-kinase (PI3K) involvement in cancer development, there is limited information about the role of PI3K regulatory subunits. PIK3R3, the gene encodes the PI3K regulatory subunit p55 gamma, is over-expressed in glioblastoma and ovarian cancers, but its expression in gastric cancer (GC) is not known. We thus used genetic and bioinformatic approaches to examine PIK3R3 expression and function in GC, the second leading cause of cancer mortality world-wide and highly prevalent among Asians. METHODS: Primary GC and matched non-neoplastic mucosa tissue specimens from a unique Asian patient gastric cancer library were comprehensively profiled with platforms that measured genome-wide mRNA expression, DNA copy number variation, and DNA methylation status. Function of PIK3R3 was predicted by IPA pathway analysis of co-regulated genes with PIK3R3, and further investigated by siRNA knockdown studies. Cell proliferation was estimated by crystal violet dye elution and BrdU incorporation assay. Cell cycle distribution was analysed by FACS. RESULTS: PIK3R3 was significantly up-regulated in GC specimens (n=126, p<0.05), and 9.5 to 15% tumors showed more than 2 fold increase compare to the paired mucosa tissues. IPA pathway analysis showed that PIK3R3 promoted cellular growth and proliferation. Knockdown of PIK3R3 decreased the growth of GC cells, induced G0/G1 cell cycle arrest, decreased retinoblastoma protein (Rb) phosphorylation, cyclin D1, and PCNA expression. CONCLUSION: Using a combination of genetic, bioinformatic, and molecular biological approaches, we showed that PIK3R3 was up-regulated in GC and promoted cell cycle progression and proliferation; and thus may be a potential new therapeutic target for GC.     

Evaluation of Phosphatidylinositol-4-Kinase IIIα as a Hepatitis C Virus Drug Target

Phosphatidylinositol-4-kinase IIIα (PI4KIIIα) is an essential host cell factor for hepatitis C virus (HCV) replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for small-molecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIIIα inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIIIα inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4IIIα-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4IIIα inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIIIα as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIIIα, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIIIα inhibitors for treatment of chronic HCV infection.     

Somatic Mutations of PIK3R1 Promote Gliomagenesis

The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.     

Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex

Internalization of beta-adrenergic receptors (betaARs) occurs by the sequential binding of beta-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that betaARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from betaARK1 and prevents betaARK1-mediated translocation of PI3K to activated beta2ARs. Furthermore, disruption of the betaARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the beta2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to betaARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.     

Local change in phospholipid composition at the cleavage furrow is essential for completion of cytokinesis

Cell division ends up with the membrane separation of two daughter cells, presumably by a membrane fusion that requires dynamic changes of the distribution and the composition of membrane lipids. We have previously shown that a membrane lipid phosphatidylethanolamine (PE) is exposed on the cell surface of the cleavage furrow during late cytokinesis and that this PE movement is involved in regulation of the contractile ring disassembly. Here we show that immobilization of cell surface PE by a PE-binding peptide blocks the RhoA inactivation in the late stage of cytokinesis. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), but not other RhoA effectors, is co-localized with RhoA in the peptide-treated cells. Indeed, PIP5K and its product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are localized to the cleavage furrow of normally dividing cells. Both overexpression of a kinase-deficient PIP5K mutant and microinjection of anti-PI(4,5)P(2) antibodies compromise cytokinesis by preventing local accumulation of PI(4,5)P(2) in the cleavage furrow. These findings demonstrate that the localized production of PI(4,5)P(2) is required for the proper completion of cytokinesis and that the possible formation of a unique lipid domain in the cleavage furrow membrane may play a crucial role in coordinating the contractile rearrangement with the membrane remodeling during late cytokinesis.     

Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts.

We investigated the extent to which phosphatidylinositol 3-kinase (PI 3-kinase) and Rac, a member of the Rho family of small GTPases, are involved in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha leading to activation of c-fos serum response element (SRE) and c-Jun amino-terminal kinase (JNK) in Rat-2 fibroblasts. Inhibition of PI 3-kinase by LY294002 or wortmannin, two specific PI 3-kinase antagonists, or co-transfection with a dominant negative mutant of PI 3-kinase dose-dependently blocked stimulation of c-fos SRE by TNF-alpha. Similarly, LY294002 significantly diminished TNF-alpha-induced activation of JNK, suggesting that nuclear signaling triggered by TNF-alpha is dependent on PI 3-kinase-mediated activation of both c-fos SRE and JNK. We also found nuclear signaling by TNF-alpha to be Rac-dependent, as demonstrated by the inhibitory effect of transient co-transfection with a dominant negative Rac mutant, RacN17. Our findings suggest that Rac is situated downstream of PI 3-kinase in the TNF-alpha signaling pathway to the nucleus, and we conclude that PI 3-kinase and Rac each plays a pivotal role in the nuclear signaling cascade triggered by TNF-alpha.     

PIKE GTPase-mediated nuclear signalings promote cell survival

The nuclear GTPase PIKE (PI 3-kinase Enhancer) binds PI 3-kinase and enhances it lipid kinase activity. PIKE predominantly distributes in the brain, and nerve growth factor stimulation triggers PIKE activation by provoking nuclear translocation of PLC-gamma1, which acts as a physiologic guanine nucleotide exchange factor (GEF) for PIKE through its SH3 domain. PIKE contains GTPase and ArfGAP domains, which are separated by a PH domain. C-terminal ArfGAP domain activates its internal GTPase activity, and this process is regulated by the interaction between phosphatidylinositols and PH domain. PI 3-kinase occurs in the nuclei of a broad range of cell types, and various stimuli elicit its nuclear translocation. The nuclei from NGF-treated PC12 cells are resistant to DNA fragmentation initiated by activated cell-free apoptosome, for which PIKE/nuclear PI 3-kinase signaling through nuclear PI(3,4,5)P(3) and Akt plays an essential role. As a nuclear receptor for PI(3,4,5)P(3,) B23 binds to PI(3,4,5)P(3) in an NGF-dependent way. The PI(3,4,5)P(3)/B23 complex inhibits DNA fragmentation activity of CAD. Nuclear Akt regulation of apoptosis is dependent on its phosphorylation of key substrates in the nucleus, but the identities of these substrates are unknown. Identification of its nuclear substrates will further our understanding of the physiological roles of nuclear PI 3-kinase/Akt signaling.     

Phosphatidylinositol 4-kinase III beta is a target of enviroxime-like compounds for antipoliovirus activity

Enviroxime is an antienterovirus compound that targets viral protein 3A and/or 3AB and suppresses a step in enterovirus replication by unknown mechanism. To date, four antienterovirus compounds, i.e., GW5074, Flt3 inhibitor II, TTP-8307, and AN-12-H5, are known to have similar mutations in the 3A protein-encoding region causing resistance to enviroxime (a G5318A [3A-Ala70Thr] mutation in poliovirus [PV]) and are considered enviroxime-like compounds. Recently, antienterovirus activity of a phosphatidylinositol 4-kinase III beta (PI4KB) inhibitor, PIK93, was reported, suggesting that PI4KB is an important host factor targetable by antienterovirus compounds (N. Y. Hsu et al., Cell 141:799-811, 2010). In this study, we analyzed the inhibitory effects of previously identified enviroxime-like compounds (GW5074 and AN-12-H5) and a newly identified antienterovirus compound, T-00127-HEV1, on phosphoinositide (PI) kinases. We found that T-00127-HEV1 inhibited PI4KB activity with a higher specificity for than other PI kinases, in contrast to GW5074, which had a broad specificity for PI kinases. In contrast, AN-12-H5 showed no inhibitory effect on PI4KB activity and only moderate inhibitory effects on PI 3-kinase activity. Small interfering RNA (siRNA) screening targeting PI kinases identified PI4KB is a target of GW5074 and T-00127-HEV1, but not of AN-12-H5, for anti-PV activity. Interestingly, T-00127-HEV1 and GW5074 did not inhibit hepatitis C virus (HCV) replication, in contrast to a strong inhibitory effect of AN-12-H5. These results suggested that PI4KB is an enterovirus-specific host factor required for the replication process and targeted by some enviroxime-like compounds (T-00127-HEV1 and GW5074) and that enviroxime-like compounds may have targets other than PI kinases for their antiviral effect.