Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53-null tumors.

Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or ß (PI5P4Kα and ß) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and ß in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K ß and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A(-/-), PIP4K2B(+/-), and TP53(-/-) mice were viable and had a dramatic reduction in tumor formation compared to TP53(-/-) littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.

Phosphoinositide signalling in type 2 diabetes: a ß-cell perspective.

Type 2 diabetes is a complex disease. It results from a failure of the body to maintain energy homoeostasis. Multicellular organisms have evolved complex strategies to preserve a relatively stable internal nutrient environment, despite fluctuations in external nutrient availability. This complex strategy involves the co-ordinated responses of multiple organs to promote storage or mobilization of energy sources according to the availability of nutrients and cellular bioenergetics needs. The endocrine pancreas plays a central role in these processes by secreting insulin and glucagon. When this co-ordinated effort fails, hyperglycaemia and hyperlipidaemia develops, characterizing a state of metabolic imbalance and ultimately overt diabetes. Although diabetes is most likely a collection of diseases, scientists are starting to identify genetic components and environmental triggers. Genome-wide association studies revealed that by and large, gene variants associated with type 2 diabetes are implicated in pancreatic ß-cell function, suggesting that the ß-cell may be the weakest link in the chain of events that results in diabetes. Thus, it is critical to understand how environmental cues affect the ß-cell. Phosphoinositides are important 'decoders' of environmental cues. As such, these lipids have been implicated in cellular responses to a wide range of growth factors, hormones, stress agents, nutrients and metabolites. Here we will review some of the well-established and potential new roles for phosphoinositides in ß-cell function/dysfunction and discuss how our knowledge of phosphoinositide signalling could aid in the identification of potential strategies for treating or preventing type 2 diabetes.

Production of phosphatidylinositol 5-phosphate via PIKfyve and MTMR3 regulates cell migration.

Although phosphatidylinositol 5-phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3-kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P-binding motifs, we identified the phosphoinositide 5-kinase PIKfyve and the phosphoinositide 3-phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P(2). The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P-producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.

Multiple inositol phosphate species enhance stability of active mTOR.

Mechanistic Target of Rapamycin (mTOR) binds the small metabolite inositol hexakisphosphate (IP6) as shown in structures of mTOR, however it remains unclear if IP6, or any other inositol phosphate species, can activate mTOR kinase activity. Here, we show that multiple, exogenously added inositol phosphate species (IP6, IP5, IP4 and IP3) can all enhance the ability of mTOR and mTORC1 to auto-phosphorylate and incorporate radiolabeled phosphate into peptide substrates in in vitro kinase reactions. Although IP6 did not affect the apparent KM of mTORC1 for ATP, monitoring kinase activity over longer reaction times showed increased product formation, suggesting inositol phosphates stabilize an active form of mTORC1 in vitro. The effects of IP6 on mTOR were reversible, suggesting IP6 bound to mTOR can be exchanged dynamically with the free solvent. Interestingly, we also observed that IP6 could alter mTOR solubility and electrophoretic mobility in SDS-PAGE in the presence of manganese, suggesting divalent cations may play a role in inositol phosphate regulation of mTOR. Together, these data suggest for the first time that multiple inositol phosphate species (IP4, IP5 and IP6) can dynamically regulate mTOR and mTORC1 by promoting a stable, active state of the kinase. Our data suggest that studies of the dynamics of inositol phosphate regulation of mTOR are well justified.

Design, synthesis and cellular characterization of a new class of IPMK kinase inhibitors.

Many genetic studies have established the kinase activity of inositol phosphate multikinase (IPMK) is required for the synthesis of higher-order inositol phosphate signaling molecules, the regulation of gene expression and control of the cell cycle. These genetic studies await orthogonal validation by specific IPMK inhibitors, but no such inhibitors have been synthesized. Here, we report complete chemical synthesis, cellular characterization, structure-activity relationships and rodent pharmacokinetics of a novel series of highly potent IPMK inhibitors. The first-generation compound 1 (UNC7437) decreased cellular proliferation and tritiated inositol phosphate levels in metabolically labeled human U251-MG glioblastoma cells. Compound 1 also regulated the transcriptome of these cells, selectively regulating genes that are enriched in cancer, inflammatory and viral infection pathways. Further optimization of compound 1 eventually led to compound 15 (UNC9750), which showed improved potency and pharmacokinetics in rodents. Compound 15 specifically inhibited cellular accumulation of InsP 5 , a direct product of IPMK kinase activity, while having no effect on InsP 6 levels, revealing a novel metabolic signature detected for the first time by rapid chemical attenuation of cellular IPMK activity. These studies designed, optimized and synthesized a new series of IPMK inhibitors, which reduces glioblastoma cell growth, induces a novel InsP 5 metabolic signature, and reveals novel aspects inositol phosphate cellular metabolism and signaling.

IPMK regulates HDAC3 activity and histone H4 acetylation in human cells.

Histone deacetylases (HDACs) repress transcription by catalyzing the removal of acetyl groups from histones. Class 1 HDACs are activated by inositol phosphate signaling molecules in vitro , but it is unclear if this regulation occurs in human cells. Inositol Polyphosphate Multikinase (IPMK) is required for production of inositol hexakisphosphate (IP6), pentakisphosphate (IP5) and certain tetrakisphosphate (IP4) species, all known activators of Class 1 HDACs in vitro . Here, we generated IPMK knockout (IKO) human U251 glioblastoma cells, which decreased cellular inositol phosphate levels and increased histone H4-acetylation by mass spectrometry. ChIP-seq showed IKO increased H4-acetylation at IKO-upregulated genes, but H4-acetylation was unchanged at IKO-downregulated genes, suggesting gene-specific responses to IPMK knockout. HDAC deacetylase enzyme activity was decreased in HDAC3 immunoprecipitates from IKO vs . wild-type cells, while deacetylase activity of other Class 1 HDACs had no detectable changes in activity. Wild-type IPMK expression in IKO cells fully rescued HDAC3 deacetylase activity, while kinase-dead IPMK expression had no effect. Further, the deficiency in HDAC3 activity in immunoprecipitates from IKO cells could be fully rescued by addition of synthesized IP4 (Ins(1,4,5,6)P4) to the enzyme assay, while control inositol had no effect. These data suggest that cellular IPMK-dependent inositol phosphates are required for full HDAC3 enzyme activity and proper histone H4-acetylation. Implications for targeting IPMK in HDAC3-dependent diseases are discussed.

25 Years of PI5P.

The accidental discovery of PI5P (phosphatidylinositol-5-phosphate) was published 25 years ago, when PIP5K type II (phosphoinositide-4-phosphate 5-kinase) was shown to actually be a 4-kinase that uses PI5P as a substrate to generate PI(4,5)P2. Consequently, PIP5K type II was renamed to PI5P4K, or PIP4K for short, and PI5P became the last of the 7 signaling phosphoinositides to be discovered. Much of what we know about PI5P comes from genetic studies of PIP4K, as the pathways for PI5P synthesis, the downstream targets of PI5P and how PI5P affects cellular function all remain largely enigmatic. Nevertheless, PI5P and PI5P-dependent PI(4,5)P2 synthesis have been clearly implicated in metabolic homeostasis and in diseases such as cancer. Here, we review the past 25 years of PI5P research, with particular emphasis on the impact this small signaling lipid has on human health.

Mitochondrial transporter expression patterns distinguish tumor from normal tissue and identify cancer subtypes with different survival and metabolism.

Transporters of the inner mitochondrial membrane are essential to metabolism. We demonstrate that metabolism as represented by expression of genes encoding SLC25 transporters differentiates human cancers. Tumor to normal tissue expression ratios for clear cell renal cell carcinoma, colon adenocarcinoma, lung adenocarcinoma and breast invasive carcinoma were found to be highly significant. Affinity propagation trained on SLC25 gene expression patterns from 19 human cancer types (6825 TCGA samples) and normal tissues (2322 GTEx samples) was used to generate clusters. They differentiate cancers from normal tissues. They also indicate cancer subtypes with survivals distinct from the total patient population of the cancer type. Probing the kidney, colon, lung, and breast cancer clusters, subtype pairs of cancers were identified with distinct prognoses and differing in expression of protein coding genes from among 2080 metabolic enzymes assayed. We demonstrate that SLC25 expression clusters facilitate the identification of the tissue-of-origin, essential to efficacy of most cancer therapies, of CUPs (cancer-unknown-primary) known to have poor prognoses. Different cancer types within a single cluster have similar metabolic patterns and this raises the possibility that such cancers may respond similarly to existing and new anti-cancer therapies.

A novel HPLC-based approach makes possible the spatial characterization of cellular PtdIns5P and other phosphoinositides.

PtdIns5P was discovered in 1997 [Rameh, Tolias, Duckworth and Cantley (1997) Nature 390, 192-196], but still very little is known about its regulation and function. Hitherto, studies of PtdIns5P regulation have been hindered by the inability to measure cellular PtdIns5P using conventional HPLC, owing to poor separation from PtdIns4P. In the present paper we describe a new HPLC method for resolving PtdIns5P from PtdIns4P, which makes possible accurate measurements of basal and inducible levels of cellular PtdIns5P in the context of other phosphoinositides. Using this new method, we found that PtdIns5P is constitutively present in all cells examined (epithelial cells, fibroblasts and myoblasts, among others) at levels typically 1-2% of PtdIns4P levels. In the beta-pancreatic cell line BTC6, which is specialized in insulin secretion, PtdIns5P levels were higher than in most cells (2.5-4% of PtdIns4P). Using subcellular fractionation, we found that the majority of the basal PtdIns5P is present in the plasma membrane, but it is also enriched in intracellular membrane compartments, especially in SER (smooth endoplasmic reticulum) and/or Golgi, where high levels of PtdIns3P were also detected. Unlike PtdIns3P, PtdIns5P was also found in fractions containing very-low-density vesicles. Knockdown of PIP4K (PtdIns5P 4-kinase) leads to accumulation of PtdIns5P in light fractions and fractions enriched in SER/Golgi, whereas treatment with Brefeldin A results in a subtle, but reproducible, change in PtdIns5P distribution. These results indicate that basal PtdIns5P and the PtdIns5P pathway for PtdIns(4,5)P(2) synthesis may play a role in Golgi-mediated vesicle trafficking.

Lipid research picks up speed on the slopes of Taos.

The 2002 Keystone Symposium on "Regulation of Cellular Responses by Lipid Mediators" provided a lively and active forum to discuss research in lipid signaling. This meeting review can provide only a glimpse into the diversity of research presented. Here we have chosen to highlight a group of exciting presentations describing novel features of the temporal and spatial regulation of phosphoinositides and their downstream targets.

A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast.

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.

Increased insulin sensitivity and reduced adiposity in phosphatidylinositol 5-phosphate 4-kinase beta-/- mice.

Phosphorylated derivatives of the lipid phosphatidylinositol are known to play critical roles in insulin response. Phosphatidylinositol 5-phosphate 4-kinases convert phosphatidylinositol 5-phosphate to phosphatidylinositol 4,5-bis-phosphate. To understand the physiological role of these kinases, we generated mice that do not express phosphatidylinositol 5-phosphate 4-kinase beta. These mice are hypersensitive to insulin and have reduced body weights compared to wild-type littermates. While adult male mice lacking phosphatidylinositol 5-phosphate 4-kinase beta have significantly less body fat than wild-type littermates, female mice lacking phosphatidylinositol 5-phosphate 4-kinase beta have increased insulin sensitivity in the presence of normal adiposity. Furthermore, in vivo insulin-induced activation of the protein kinase Akt is enhanced in skeletal muscle and liver from mice lacking phosphatidylinositol 5-phosphate 4-kinase beta. These results indicate that phosphatidylinositol 5-phosphate 4-kinase beta plays a role in determining insulin sensitivity and adiposity in vivo and suggest that inhibitors of this enzyme may be useful in the treatment of type 2 diabetes.

Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.

Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.

Analysis of the Phosphoinositide Composition of Subcellular Membrane Fractions.

Phosphoinositides play critical roles in the transduction of extracellular signals through the plasma membrane and also in endomembrane events important for vesicle trafficking and organelle function (Di Paolo and De Camilli, Nature 443(7112):651-657, 2006). The response triggered by these lipids is heavily dependent on the microenvironment in which they are found. HPLC analysis of labeled phosphoinositides allows quantification of the levels of each phosphoinositide species relative to their precursor, phosphatidylinositol. When combined with subcellular fractionation techniques, this strategy allows measurement of the relative phosphoinositide composition of each membrane fraction or organelle and determination of the microenvironment in which each species is enriched. Here, we describe the steps to separate and quantify total or localized phosphoinositides from cultured cells.

IQGAP1 makes PI(3)K signalling as easy as PIP, PIP2, PIP3.

Despite being one of the most studied signalling pathways, precisely how phospholipid synthesis is regulated in the phosphoinositide signalling cascade remains unclear. The scaffold protein IQGAP1 is now shown to orchestrate the assembly of a multi-enzyme complex that streamlines PtdIns(3,4,5)P3 synthesis to facilitate Akt activation in response to extracellular stimuli.

PIP4kγ is a substrate for mTORC1 that maintains basal mTORC1 signaling during starvation.

Phosphatidylinositol-5-phosphate 4-kinases (PIP4ks) are a family of lipid kinases that specifically use phosphatidylinositol 5-monophosphate (PI-5-P) as a substrate to synthesize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Suppression of PIP4k function in Drosophila results in smaller cells and reduced target of rapamycin complex 1 (TORC1) signaling. We showed that the γ isoform of PIP4k stimulated signaling through mammalian TORC1 (mTORC1). Knockdown of PIP4kγ reduced cell mass in cells in which mTORC1 is constitutively activated by Tsc2 deficiency. In Tsc2 null cells, mTORC1 activation was partially independent of amino acids or glucose and glutamine. PIP4kγ knockdown inhibited the nutrient-independent activation of mTORC1 in Tsc2 knockdown cells and reduced basal mTORC1 signaling in wild-type cells. PIP4kγ was phosphorylated by mTORC1 and associated with the complex. Phosphorylated PIP4kγ was enriched in light microsomal vesicles, whereas the unphosphorylated form was enriched in heavy microsomal vesicles associated with the Golgi. Furthermore, basal mTORC1 signaling was enhanced by overexpression of unphosphorylated wild-type PIP4kγ or a phosphorylation-defective mutant and decreased by overexpression of a phosphorylation-mimetic mutant. Together, these results demonstrate that PIP4kγ and mTORC1 interact in a self-regulated feedback loop to maintain low and tightly regulated mTORC1 activation during starvation.

AKT facilitates EGFR trafficking and degradation by phosphorylating and activating PIKfyve.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that controls cell proliferation, growth, survival, metabolism, and migration by activating the PI3K (phosphatidylinositol 3-kinase)-AKT and ERK (extracellular signal-regulated kinase)-RSK (ribosomal S6 kinase) pathways. EGFR signaling to these pathways is temporally and spatially regulated. Endocytic trafficking controls the access of EGFR to these downstream effectors and also its degradation, which terminates EGFR signaling. We showed that AKT facilitated the endocytic trafficking of EGFR to promote its degradation. Interfering with AKT signaling reduced both EGFR recycling and the rate of EGFR degradation. In AKT-impaired cells, EGFRs were unable to reach the cell surface or the lysosomal compartment and accumulated in the early endosomes, resulting in prolonged signaling and increased activation of ERK and RSK. Upon EGF stimulation, AKT phosphorylated and activated the kinase PIKfyve [FYVE-containing phosphatidylinositol 3-phosphate 5-kinase], which promoted vesicle trafficking to lysosomes. PIKfyve activation promoted EGFR degradation. Similar regulation occurred with platelet-derived growth factor receptor (PDGFR), suggesting that AKT phosphorylation and activation of PIKfyve is likely to be a common feedback mechanism for terminating RTK signaling and reducing receptor abundance.

AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer.

Dysregulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway occurs frequently in human cancer. PTEN tumor suppressor or PIK3CA oncogene mutations both direct PI3K-dependent tumorigenesis largely through activation of the AKT/PKB kinase. However, here we show through phosphoprotein profiling and functional genomic studies that many PIK3CA mutant cancer cell lines and human breast tumors exhibit only minimal AKT activation and a diminished reliance on AKT for anchorage-independent growth. Instead, these cells retain robust PDK1 activation and membrane localization and exhibit dependency on the PDK1 substrate SGK3. SGK3 undergoes PI3K- and PDK1-dependent activation in PIK3CA mutant cancer cells. Thus, PI3K may promote cancer through both AKT-dependent and AKT-independent mechanisms. Knowledge of differential PI3K/PDK1 signaling could inform rational therapeutics in cancers harboring PIK3CA mutations.

SopB promotes phosphatidylinositol 3-phosphate formation on Salmonella vacuoles by recruiting Rab5 and Vps34.

Salmonella colonizes a vacuolar niche in host cells during infection. Maturation of the Salmonella-containing vacuole (SCV) involves the formation of phosphatidylinositol 3-phosphate (PI(3)P) on its outer leaflet. SopB, a bacterial virulence factor with phosphoinositide phosphatase activity, was proposed to generate PI(3)P by dephosphorylating PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3. Here, we examine the mechanism of PI(3)P formation during Salmonella infection. SopB is required to form PI(3,4)P2/PI(3,4,5)P3 at invasion ruffles and PI(3)P on nascent SCVs. However, we uncouple these events experimentally and reveal that SopB does not dephosphorylate PI(3,4)P2/PI(3,4,5)P3 to produce PI(3)P. Instead, the phosphatase activity of SopB is required for Rab5 recruitment to the SCV. Vps34, a PI3-kinase that associates with active Rab5, is responsible for PI(3)P formation on SCVs. Therefore, SopB mediates PI(3)P production on the SCV indirectly through recruitment of Rab5 and its effector Vps34. These findings reveal a link between phosphoinositide phosphatase activity and the recruitment of Rab5 to phagosomes.

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).