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Autotaxin (ATX) is a secreted glycoprotein and the only member of the ectonucleotide pyrophosphatase/phosphodiesterase family that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key responses, such as cell migration, proliferation, and survival, implicating ATX-LPA signaling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, called the "pocket" and the "tunnel," respectively. However, the mechanisms in allosteric modulation of ATX's activity as a lysophospholipase D are unclear. Here, using the physiological LPC substrate, a new fluorescent substrate, and diverse ATX inhibitors, we revisited the kinetics and allosteric regulation of the ATX catalytic cycle, dissecting the different steps and pathways leading to LPC hydrolysis. We found that ATX activity is stimulated by LPA and that LPA activates ATX lysophospholipase D activity by binding to the ATX tunnel. A consolidation of all experimental kinetics data yielded a comprehensive catalytic model supported by molecular modeling simulations and suggested a positive feedback mechanism that is regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend the current understanding of ATX hydrolysis in light of the allosteric regulation by ATX-produced LPA species and have implications for the design and application of both orthosteric and allosteric ATX inhibitors.

Glucose provides an essential nutrient source that supports glycolysis and the hexosamine biosynthesis pathway (HBP) to maintain tumour cell growth and survival. Here we investigated if short-term glucose deprivation specifically modulates the phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) cell survival pathway. Insulin-stimulated PKB activation was strongly abrogated in the absence of extracellular glucose as a consequence of the loss of insulin-stimulated PI3K activation and short-term glucose deprivation inhibited subsequent tumour cell growth. Loss of insulin-stimulated PKB signalling and cell growth was rescued by extracellular glucosamine and increased flux through the HBP. Disruption of O-GlcNAc transferase activity, a terminal step in the HBP, implicated O-GlcNAcylation in PKB signalling and cell growth. Glycogenolysis is known to support cell survival during glucose deprivation, and in A549 lung cancer cells its inhibition attenuates PKB activation which is rescued by increased flux through the HBP. Our studies show that rerouting of glycolytic metabolites to the HBP under glucose-restricted conditions maintains PI3K/PKB signalling enabling cell survival and proliferation.

Phosphatidylinositol-5-phosphate (PtdIns5P) 4-kinase β (PIP4K2B) directly regulates the levels of two important phosphoinositide second messengers, PtdIns5P and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2]. PIP4K2B has been linked to the regulation of gene transcription, to TP53 and AKT activation, and to the regulation of cellular reactive oxygen accumulation. However, its role in human tumor development and on patient survival is not known. Here, we have interrogated the expression of PIP4K2B in a cohort (489) of patients with breast tumor using immunohistochemical staining and by a meta-analysis of gene expression profiles from 2,999 breast tumors, both with associated clinical outcome data. Low PIP4K2B expression was associated with increased tumor size, high Nottingham histological grade, Ki67 expression, and distant metastasis, whereas high PIP4K2B expression strongly associated with ERBB2 expression. Kaplan–Meier curves showed that both high and low PIP4K2B expression correlated with poorer patient survival compared with intermediate expression. In normal (MCF10A) and tumor (MCF7) breast epithelial cell lines, mimicking low PIP4K2B expression, using short hairpin RNA interference-mediated knockdown, led to a decrease in the transcription and expression of the tumor suppressor protein E-cadherin (CDH1). In MCF10A cells, knockdown of PIP4K2B enhanced TGF-β–induced epithelial to mesenchymal transition (EMT), a process required during the development of metastasis. Analysis of gene expression datasets confirmed the association between low PIP4K2B and low CDH1expression. Decreased CDH1 expression and enhancement of TGF-β–induced EMT by reduced PIP4K2B expression might, in part, explain the association between low PIP4K2B expression and poor patient survival. Cancer Res; 73(23); 6913–25. ©2013 AACR.

Autotaxin (ATX) is a secreted phosphodiesterase that produces the signalling lipid lysophosphatidic acid (LPA). The bimetallic active site of ATX is structurally related to the alkaline phosphatase superfamily. Here, we present a new crystal structure of ATX in complex with orthovanadate (ATX-VO5), which binds the Oγ nucleophile of Thr209 and adopts a trigonal bipyramidal conformation, following the nucleophile attack onto the substrate. We have now a portfolio of ATX structures we discuss as intermediates of the catalytic mechanism: the new ATX-VO5 structure; a unique structure where the nucleophile Thr209 is phosphorylated (ATX-pThr). Comparing these to a complex with the LPA product (ATX-LPA) and with a complex with a phosphate ion (ATX-PO4), that represent the Michaelis complex of the reaction, we observe movements of Thr209, changes in the relative displacement of the zinc ions, and a water molecule that likely fulfils the second nucleophilic attack. We propose that ATX follows the associative two-step in-line displacement mechanism.

Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs., Autotaxin generates the bioactive lipid lysophosphatidic acid to regulate diverse biological processes. Here, the authors identify a role for bile salts as direct allosteric inhibitors of autotaxin activity, suggesting that steroids may function as regulators of lysophosphatidic acid signalling.

Background: Transcriptionally active high-risk human papilloma viruses (HPVs), particularly HPV type 16 (HPV16), are found in a subset of head and neck squamous-cell carcinomas (HNSCCs). HPV16-associated carcinogenesis is mediated by expression of the viral E6 and E7 oncoproteins, which cause deregulation of the cell cycle by inactivating p53 and pRb, respectively. We tested the hypothesis that HPVassociated HNSCCs display a pattern of genetic alterations different from those of HNSCCs without HPV DNA. Methods: Polymerase chain reaction‐based assays were used to examine 143 consecutive HNSCCs (106 of the oral cavity and 37 of the oropharynx) for the presence of HPV DNA and for viral E6 and/or E7 messenger RNA (mRNA) expression. The HPV DNA‐and E6 and E7 mRNA‐positive HNSCCs and an equal number of HPV DNA‐negative HNSCCs were further analyzed for mutations in TP53, the gene encoding p53, and for allelic loss of 28 microsatellite markers at chromosome arms 3p, 6q, 8p, 9p, 13q, 17p, and 18q, including markers located in regions of chromosome arms 9p and 17p that harbor genes involved the p53 and pRb pathways. All statistical tests were two-sided. Results: Twenty-four (16.7%) of the 143 HNSCCs were positive for HPV16 DNA, and 12 of these HNSCCs (8.4% of total number) expressed E6 and E7 mRNAs. None of the HPV DNA‐and E6/E7 mRNA‐positive tumors had TP53 gene mutations, whereas nine (75%) of the 12 HPV DNA‐negative tumors had such mutations (P

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated lipid kinases that phosphorylate PtdIns5P to generate PtdIns(4,5)P2. There are three isoforms of PIP4Ks: PIP4K2A, 2B and 2C, which localise to different subcellular compartments with the PIP4K2B isoform being localised predominantly in the nucleus. Suppression of PIP4K expression selectively prevents tumour cell growth in vitro and prevents tumour development in mice that have lost the tumour suppressor p53. p53 is lost or mutated in over 70% of all human tumours. These studies suggest that inhibition of PIP4K signalling constitutes a novel anti-cancer therapeutic target. In this review we will discuss the role of PIP4K in tumour suppression and speculate on how PIP4K modulates nuclear phosphoinositides (PPIns) and how this might impact on nuclear functions to regulate cell growth. This article is part of a Special Issue entitled Phosphoinositides.

The spatial and temporal regulation of the second messenger PtdIns(4,5) P 2 has been shown to be crucial for regulating numerous processes in the cytoplasm and in the nucleus. Three isoforms of PIP5K1 (phosphatidylinositol 4-phosphate 5-kinase), A, B and C, are responsible for the regulation of the major pools of cellular PtdIns(4,5) P 2 . PIP5K1B is negatively regulated in response to oxidative stress although it remains unclear which pathways regulate its activity. In the present study, we have investigated the regulation of PIP5K1B by protein phosphorylation. Using MS analysis, we identified 12 phosphorylation sites on PIP5K1B. We developed a phospho-specific antibody against Ser 413 and showed that its phosphorylation was increased in response to treatment of cells with phorbol ester, H 2 O 2 or energy restriction. Using inhibitors, we define a stress-dependent pathway that requires the activity of the cellular energy sensor AMPK (AMP-activated protein kinase) and PKC (protein kinase C) to regulate Ser 413 phosphorylation. Furthermore, we demonstrate that PKC can directly phosphorylate Ser 413 in vitro . Mutation of Ser 413 to aspartate to mimic serine phosphorylation decreased both PIP5K1B activity in vitro and PtdIns(4,5) P 2 synthesis in vivo . Our studies show that collaboration between AMPK and PKC dictates the extent of Ser 413 phosphorylation on PIP5K1B and regulates PtdIns(4,5) P 2 synthesis.

Oxidative signaling is important in cellular health, involved in aging and contributes to the development of several diseases such as cancer, neurodegeneration and diabetes. Correct management of reactive oxygen species (ROS) prevents oxidative stress within cells and is imperative for cellular wellbeing. A key pathway that is regulated by oxidative stress is the activation of proline-directed stress kinases (p38, JNK). Phosphorylation induced by these kinases is often translated into cellular outcome through the recruitment of the prolyl-isomerase Pin1. Pin1 binds to phosphorylated substrates using its WW-domain and can induce conformational changes in the target protein through its prolyl-isomerase activity. We show that exposure of cells to UV irradiation or hydrogen peroxide (H₂O₂), induces the synthesis of the phosphoinositide second messenger PtdIns5P in part by inducing the interaction between phosphatidylinositol-5-phosphate 4-kinase (PIP4K) enzymes that remove PtdIns5P, with Pin1. In response to H₂O₂ exposure, Murine Embryonic Fibroblasts (MEFs) derived from Pin1⁻/⁻ mice showed increased cell viability and an increased abundance of PtdIns5P compared to wild-type MEFs. Decreasing the levels of PtdIns5P in Pin1⁻/⁻ MEFs decreased both their viability in response to H₂O₂ exposure and the expression of genes required for cellular ROS management. The decrease in the expression of these genes manifested itself in the increased accumulation of cellular ROS. These data strongly argue that PtdIns5P acts as a stress-induced second messenger that can calibrate how cells manage ROS.

Lipid signalling in human disease is an important field of investigation and stems from the fact that phosphoinositide signalling has been implicated in the control of nearly all the important cellular pathways including metabolism, cell cycle control, membrane trafficking, apoptosis and neuronal conduction. A distinct nuclear inositide signalling metabolism has been identified, thus defining a new role for inositides in the nucleus, which are now considered essential co-factors for several nuclear processes, including DNA repair, transcription regulation, and RNA dynamics. Deregulation of phoshoinositide metabolism within the nuclear compartment may contribute to disease progression in several disorders, such as chronic inflammation, cancer, metabolic, and degenerative syndromes. In order to utilize these very druggable pathways for human benefit there is a need to identify how nuclear inositides are regulated specifically within this compartment and what downstream nuclear effectors process and integrate inositide signalling cascades in order to specifically control nuclear function. Here we describe some of the facets of nuclear inositide metabolism with a focus on their relationship to cell cycle control and differentiation.

The beta-isoform of PIP4K (PtdIns5P-4-kinase) regulates the levels of nuclear PtdIns5P, which in turn modulates the acetylation of the tumour suppressor p53. The crystal structure of PIP4Kbeta demonstrated that it can form a homodimer with the two subunits arranged in opposite orientations. Using MS, isoform-specific antibodies against PIP4Ks, RNAi (RNA interference) suppression and overexpression studies, we show that PIP4Kbeta interacts in vitro and in vivo with the PIP4Kalpha isoform. As the two isoforms phosphorylate the same substrate to generate the same product, the interaction could be considered to be functionally redundant. However, contrary to expectation, we find that PIP4Kbeta has 2000-fold less activity towards PtdIns5P compared with PIP4Kalpha, and that the majority of PIP4K activity associated with PIP4Kbeta comes from its interaction with PIP4Kalpha. Furthermore, PIP4Kbeta can modulate the nuclear localization of PIP4Kalpha, and PIP4Kalpha has a role in regulating PIP4Kbeta functions. The results of the present study suggest a rationale for the functional interaction between PIP4Kalpha and PIP4Kbeta and provide insight into how the relative levels of the two enzymes may be important in their physiological and pathological roles.

Phosphatidylinositol (PtdIns) and its phosphorylated derivatives represent less than 5% of total membrane phospholipids in cells. Despite their low abundance, they form a dynamic signaling system that is regulated in response to a variety of extra- and intracellular cues. Protein domains including PH, FYVE, ENTH, PHOX, PHD fingers, and lysine-/arginine-rich patches can bind to specific phosphoinositide isomers, which, in turn, can induce changes in the subcellular localization, posttranslational modification, protein interaction partners, or activity of the protein containing such a domain. Phosphoinositides and the enzymes that synthesize them are found in many different subcellular compartments including the nuclear matrix, heterochromatin, and sites of active RNA splicing, suggesting that phosphoinositides may regulate specific functions within the nuclear compartment. The existence of distinct subcellular pools has led to the challenging task of the quantitation of temporal and spatial changes in phosphoinositides. We report methods to measure the mass levels of three different phosphoinositides within the nuclear compartment.

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.