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.
UHRF1 is a multidomain protein crucially linking histone H3 modification states and DNA methylation. While the interaction properties of its specific domains are well characterized, little is known about the regulation of these functionalities. We show that UHRF1 exists in distinct active states, binding either unmodified H3 or the H3 lysine 9 trimethylation (H3K9me3) modification. A polybasic region (PBR) in the C terminus blocks interaction of a tandem tudor domain (TTD) with H3K9me3 by occupying an essential peptide-binding groove. In this state the plant homeodomain (PHD) mediates interaction with the extreme N terminus of the unmodified H3 tail. Binding of the phosphatidylinositol phosphate PI5P to the PBR of UHRF1 results in a conformational rearrangement of the domains, allowing the TTD to bind H3K9me3. Our results define an allosteric mechanism controlling heterochromatin association of an essential regulatory protein of epigenetic states and identify a functional role for enigmatic nuclear phosphatidylinositol phosphates.
The spatial and temporal regulation of the second messenger PtdIns(4,5)P2 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)P2. 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 Ser413 and showed that its phosphorylation was increased in response to treatment of cells with phorbol ester, H2O2 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 Ser413 phosphorylation. Furthermore, we demonstrate that PKC can directly phosphorylate Ser413 in vitro. Mutation of Ser413 to aspartate to mimic serine phosphorylation decreased both PIP5K1B activity in vitro and PtdIns(4,5)P2 synthesis in vivo. Our studies show that collaboration between AMPK and PKC dictates the extent of Ser413 phosphorylation on PIP5K1B and regulates PtdIns(4,5)P2 synthesis.
Polyphosphoinositides (PPIn) are important lipid molecules whose levels are de-regulated in human diseases such as cancer, neurodegenerative disorders and metabolic syndromes. PPIn are synthesized and degraded by an array of kinases, phosphatases and lipases which are localized to various subcellular compartments and are subject to regulation in response to both extra- and intracellular cues. Changes in the activities of enzymes that metabolize PPIn lead to changes in the profiles of PPIn in various subcellular compartments. Understanding how subcellular PPIn are regulated and how they affect downstream signaling is critical to understanding their roles in human diseases. PPIn are present in the nucleus, and their levels are changed in response to various stimuli, suggesting that they may serve to regulate specific nuclear functions. However, the lack of nuclear downstream targets has hindered the definition of which pathways nuclear PPIn affect. Over recent years, targeted and global proteomic studies have identified a plethora of potential PPIn-interacting proteins involved in many aspects of transcription, chromatin remodelling and mRNA maturation, suggesting that PPIn signalling within the nucleus represents a largely unexplored novel layer of complexity in the regulation of nuclear functions.
Phosphoinositides represent a minor fraction of the total glycerolipids in cells. Despite the fact that phosphoinositides are present in small quantities, they have crucial roles during cell signaling and in regulating numerous intracellular processes. Measuring changes in the levels of different phosphoinositides in animals is difficult, but it is essential in order to define the important functions of specific members of the phosphoinositide family. Here we detail procedures for measuring phosphoinositides in 2-days-postfertilization (2-d.p.f.) embryos in zebrafish (Danio rerio). Both in vivo radiolabeling (using [(32)P]orthophosphate) followed by thin-layer or high-performance liquid chromatography (TLC or HPLC) analysis and specific in vitro phosphorylation assays (using [(32)P]?ATP) permit the quantitative measurement of phosphoinositides. Normalization of both measurements can be achieved by the determination of total lipid phosphate in embryos. All the techniques described are relatively inexpensive and accessible to most laboratories with an interest in studying the effect of gene manipulation on phosphoinositide metabolism in zebrafish. All the procedures described herein will take up to 10 working days.
During development, Drosophila larvae undergo a dramatic increase in body mass wherein nutritional and developmental cues are transduced into growth through the activity of complex signaling pathways. Class I phosphoinositide 3-kinases have an established role in this process. In this study we identify Drosophila phosphatidylinositol 5-phosphate 4-kinase (dPIP4K) as a phosphoinositide kinase that regulates growth during larval development. Loss-of-function mutants in dPIP4K show reduced body weight and prolonged larval development, whereas overexpression of dPIP4K results both in an increase in body weight and shortening of larval development. The growth defect associated with dPIP4K loss of function is accompanied by a reduction in the average cell size of larval endoreplicative tissues. Our findings reveal that these phenotypes are underpinned by changes in the signaling input into the target of rapamycin (TOR) signaling complex and changes in the activity of its direct downstream target p70 S6 kinase. Together, these results define dPIP4K activity as a regulator of cell growth and TOR signaling during larval development.
Phosphatidylinositol 5-phosphate 4-kinases (PIP4Ks) phosphorylate phosphatidylinositol 5-phosphate (PI5P) to generate phosphatidylinositol 4,5-bisphosphate; their most likely function is the regulation of the levels of PI5P, a putative signalling intermediate. There are three mammalian PIP4Ks isoforms (?, ? and ?), but their physiological roles remain poorly understood. In the present study, we identified the zebrafish orthologue (zPIP4K?) of the high-activity human PIP4K ? isoform and analyzed its role in embryonic development. RT-PCR analysis and whole-mount in situ hybridization experiments showed that zPIP4K? is maternally expressed. At later embryonic stages, high PIP4K? expression was detected in the head and the pectoral fins. Knockdown of zPIP4K? by antisense morpholino oligonucleotides led to severe morphological abnormalities, including midbody winding defects at 48hpf. The abnormal phenotype could be rescued, at least in large part, by injection of human PIP4K? mRNA. Our results reveal a key role for PIP4K? and its activity in vertebrate tissue homeostasis and organ development.
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 (H?O?), 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 H?O? 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 H?O? 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.
Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.
S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK (extracellular-signal-regulated kinase)/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, the results of the present study suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus.
Phosphoinositide 3-kinases (PI3Ks) are critical regulators of pancreatic ? cell mass and survival, whereas their involvement in insulin secretion is more controversial. Furthermore, of the different PI3Ks, the class II isoforms were detected in ? cells, although their role is still not well understood. Here we show that down-regulation of the class II PI3K isoform PI3K-C2? specifically impairs insulin granule exocytosis in rat insulinoma cells without affecting insulin content, the number of insulin granules at the plasma membrane, or the expression levels of key proteins involved in insulin secretion. Proteolysis of synaptosomal-associated protein of 25 kDa, a process involved in insulin granule exocytosis, is impaired in cells lacking PI3K-C2?. Finally, our data suggest that the mRNA for PI3K-C2? may be down-regulated in islets of Langerhans from type 2 diabetic compared with non-diabetic individuals. Our results reveal a critical role for PI3K-C2? in ? cells and suggest that down-regulation of PI3K-C2? may be a feature of type 2 diabetes.
Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(X(n= 3-7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase II?. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions.
In N1E-115 cells, neurite retraction induced by neurite remodelling factors such as lysophosphatidic acid, sphingosine 1-phosphate and semaphorin 3A require the activity of phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks). PIP5Ks synthesise the phosphoinositide lipid second messenger phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P?], and overexpression of active PIP5K is sufficient to induce neurite retraction in both N1E-115 cells and cerebellar granule neurones. However, how PIP5Ks are regulated or how they induce neurite retraction is not well defined. Here, we show that neurite retraction induced by PIP5K? is dependent on its interaction with the low molecular weight G protein Rac. We identified the interaction site between PIP5K? and Rac1 and generated a point mutant of PIP5K? that no longer interacts with endogenous Rac. Using this mutant, we show that Rac controls the plasma membrane localisation of PIP5K? and thereby the localised synthesis of PtdIns(4,5)P? required to induce neurite retraction. Mutation of this residue in other PIP5K isoforms also attenuates their ability to induce neurite retraction and to localise at the membrane. To clarify how increased levels of PtdIns(4,5)P? induce neurite retraction, we show that mutants of vinculin that are unable to interact with PtdIns(4,5)P?, attenuate PIP5K- and LPA-induced neurite retraction. Our findings support a role for PtdIns(4,5)P? synthesis in the regulation of vinculin localisation at focal complexes and ultimately in the regulation of neurite dynamics.
Phosphatidylinositol (PtdIns) and its phosphorylated derivatives represent less than 5% of total membrane phospholipids in cells. Despite their low abundance, they form a dynamic signalling system that is regulated in response to a variety of extra and intra-cellular cues (Curr Opin Genet Dev 14:196-202, 2004). Phosphoinositides and the enzymes that synthesize them are found in many different sub-cellular compartments including the nuclear matrix, heterochromatin, and sites of active RNA splicing, suggesting that phosphoinositides may regulate specific functions within the nuclear compartment (Nat Rev Mol Cell Biol 4:349-360, 2003; Curr Top Microbiol Immunol 282:177-206, 2004; Cell Mol Life Sci 61:1143-1156, 2004). The existence of distinct sub-cellular pools has led to the challenging task of understanding how the different pools are regulated and how changes in the mass of lipids within the nucleus can modulate nuclear specific pathways. Here we describe methods to determine how enzymatic activities that modulate nuclear phosphoinositides are changed in response to extracellular stimuli.
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.
Changes in gene expression enable organisms to respond to environmental stress. Levels of cellular lipid second messengers, such as the phosphoinositide PtdIns5P, change in response to a variety of stresses and can modulate the localization, conformation and activity of a number of intracellular proteins. The plant trithorax factor (ATX1) tri-methylates the lysine 4 residue of histone H3 (H3K4me3) at gene coding sequences, which positively correlates with gene transcription. Microarray analysis has identified a target gene (WRKY70) that is regulated by both ATX1 and by the exogenous addition of PtdIns5P in Arabidopsis. Interestingly, ATX1 contains a PtdIns5P interaction domain (PHD finger) and thus, phosphoinositide signaling, may link environmental stress to changes in gene transcription.
Phosphatidylinositol (4,5) bisphosphate is a lipid second messenger that controls diverse cellular processes. Phosphatidylinositolphosphate-5-kinases synthesise this lipid at the plasma membrane, although it is not clear how the localisation of these kinases is controlled. A recent study suggests that the intrinsic surface charge of the plasma membrane may be an important factor.
It has long been known that phosphoinositides are present in cellular membranes, but only in the past four decades has our understanding of their importance for proper cell function advanced significantly. Key to determining the biological roles of phosphoinositides is understanding the enzymes involved in their metabolism. Although many such enzymes have now been identified, there is still much to learn about their cellular functions. Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) are a group of kinases that catalyse the production of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)]. As well as being a substrate for the enzymes phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K), PtdIns(4,5)P(2) acts as a second messenger in its own right, influencing a variety of cellular processes. In this Commentary, we review how PIP5Ks are modulated to achieve regulated PtdIns(4,5)P(2) production, and discuss the role of these proteins in different cellular processes.
Phosphoinositides are a family of lipid second messengers interlinked by an extensive and highly regulated network of kinases and phosphatases. The modulation of phosphoinositide profiles can regulate numerous cancer-related pathways, including cell survival, cell proliferation, migration, integrin activation, and transcription. PtdIns(4,5)P2 is at the heart of phosphoinositide signaling; its levels are controlled by enzymes that synthesize it and those that degrade it. Phosphatidylinositol-4-phosphate 5-kinases (PIP5 K) phosphorylate PtdIns4P on the 5-position and constitute the major pathway for the generation of PtdIns(4,5)P2. We will discuss how to suppress the expression of human PIP5 Kbeta using RNAi and how to measure the activity and levels of the endogenous enzyme. We describe a method to immunoprecipitate the endogenous PIP5 Kbeta and to assay its activity. Western blotting with another panel of antibodies is then used to determine the levels of endogenous PIP5 Kbeta in the immunoprecipitates.
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.
Oxidative stress initiates signaling pathways, which protect from stress-induced cellular damage, initiate apoptosis, or drive cells into senescence or into tumorigenesis. Oxidative stress regulates the activity of the cell survival factor PKB, through the regulation of PtdIns(3,4,5)P? synthesis. Whether oxidative stress regulates other phosphoinositides to control PKB activation is not clear. Here we show that PtdIns5P is a redox-regulated second messenger. In response to hydrogen peroxide (H?O?), we measured an increase in PtdIns5P in cells derived from human osteosarcoma, U2OS (5-fold); breast tumors, MDA-MB-468 (2-fold); and fibrosarcoma, HT1080 (3-fold); and in p53-null murine embryonic fibroblasts (8-fold). In U2OS cells, the increase in H?O?-dependent PtdIns5P did not require mTOR, PDK1, PKB, ERK, and p38 signaling or PIKfyve, a lipid kinase that increases PtdIns5P in response to osmotic and oncogenic signaling. A reduction in H?O?-induced PtdIns5P levels by the overexpression of PIP4K revealed its role in PKB activation. Suppression of H?O?-induced PtdIns5P generation reduced PKB activation and, surprisingly, reduced cell sensitivity to growth inhibition by H?O?. These data suggest that inhibition of PIP4K signaling might be useful as a novel strategy to increase the susceptibility of tumor cells to therapeutics that function through increased oxidative stress.
At the end of cell division, cytokinesis splits the cytoplasm of nascent daughter cells and partitions segregated sister genomes. To coordinate cell division with chromosome segregation, the mitotic spindle controls cytokinetic events at the cell envelope. The spindle midzone stimulates the actomyosin-driven contraction of the cleavage furrow, which proceeds until the formation of a microtubule-rich intercellular bridge with the midbody at its centre. The midbody directs the final membrane abscission reaction and has been proposed to attach the cleavage furrow to the intercellular bridge. How the mitotic spindle is connected to the plasma membrane during cytokinesis is not understood. Here we identify a plasma membrane tethering activity in the centralspindlin protein complex, a conserved component of the spindle midzone and midbody. We demonstrate that the C1?domain of the centralspindlin subunit MgcRacGAP associates with the plasma membrane by interacting with polyanionic phosphoinositide lipids. Using X-ray crystallography we determine the structure of this atypical C1?domain. Mutations in the hydrophobic cap and in basic residues of the C1?domain of MgcRacGAP prevent association of the protein with the plasma membrane, and abrogate cytokinesis in human and chicken cells. Artificial membrane tethering of centralspindlin restores cell division in the absence of the C1?domain of MgcRacGAP. Although C1?domain function is dispensable for the formation of the midzone and midbody, it promotes contractility and is required for the attachment of the plasma membrane to the midbody, a long-postulated function of this organelle. Our analysis suggests that centralspindlin links the mitotic spindle to the plasma membrane to secure the final cut during cytokinesis in animal cells.
Oxidative signaling and oxidative stress contribute to aging, cancer, and diseases resulting from neurodegeneration. Pin1 is a proline isomerase that recognizes phosphorylated substrates and regulates the localization and conformation of its targets. Pin1(-/-) mice show phenotypes associated with premature aging, yet mouse embryonic fibroblasts (MEFs) from these mice are resistant to hydrogen peroxide (H(2)O(2))-induced cell death. We found that the abundance of phosphatidylinositol-5-phosphate (PtdIns5P) was increased in response to H(2)O(2), an effect that was enhanced in Pin1(-/-) MEFs. Reduction of H(2)O(2)-induced PtdIns5P compromised cell viability in response to oxidative stress, suggesting that PtdIns5P contributed to the enhanced cell viability of Pin1(-/-) MEFs exposed to oxidative stress. The increased PtdIns5P in the Pin1(-/-) MEFs stimulated the expression of genes involved in defense against oxidative stress and reduced the accumulation of reactive oxygen species. Pin1 and PtdIns5P 4-kinases (PIP4Ks), enzymes that phosphorylate and thereby reduce the amount of PtdIns5P, interacted in a manner dependent on the phosphorylation of PIP4K. Although reintroduction of Pin1 into the Pin1(-/-) MEFs reduced the amount of PtdIns5P produced in response to H(2)O(2), in vitro assays indicated that the isomerase activity of Pin1 inhibited PIP4K activity. Whether this isomerise-mediated inhibition of PIP4K occurs in cells remains an open question, but the data suggest that the regulation of PIP4K by Pin1 may be complex.
Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC) signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGK? isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGK? physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGK? attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGK? downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGK? translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGK? attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling.
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.
Myotubularin and myotubularin-related proteins are evolutionarily conserved in eukaryotes. Defects in their function result in muscular dystrophy, neuronal diseases and leukemia in humans. In contrast to the animal lineage, where genes encoding both active and inactive myotubularins (phosphoinositide 3-phosphatases) have appeared and proliferated in the basal metazoan group, myotubularin genes are not found in the unicellular relatives of green plants. However, they are present in land plants encoding proteins highly similar to the active metazoan enzymes. Despite their remarkable structural conservation, plant and animal myotubularins have significantly diverged in their functions. While loss of myotubularin function causes severe disease phenotypes in humans it is not essential for the cellular homeostasis under normal conditions in Arabidopsis thaliana. Instead, myotubularin deficiency is associated with altered tolerance to dehydration stress. The two Arabidopsis genes AtMTM1 and AtMTM2 have originated from a segmental chromosomal duplication and encode catalytically active enzymes. However, only AtMTM1 is involved in elevating the cellular level of phosphatidylinositol 5-phosphate in response to dehydration stress, and the two myotubularins differentially affect the Arabidopsis dehydration stress-responding transcriptome. AtMTM1 and AtMTM2 display different localization patterns in the cell, consistent with the idea that they associate with different membranes to perform specific functions. A single amino acid mutation in AtMTM2 (L250W) results in a dramatic loss of subcellular localization. Mutations in this region are linked to disease conditions in humans.
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