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Collaboration of AMPK and PKC to induce phosphorylation of Ser413 on PIP5K1B resulting in decreased kinase activity and reduced PtdIns(4,5)P2 synthesis in response to oxidative stress and energy restriction.van den Bout, Iman; Jones, David R; Shah, Zahid H; Halstead, J; Keune, Willem-Jan; Mohammed, S; D'Santos, C; Divecha, Nullin (2013-11-01)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.
Identification of nuclear phosphatidylinositol 4,5-bisphosphate-interacting proteins by neomycin extraction.Lewis, A; Sommer, Lilly; Arntzen, M; Strahm, Y; Morrice, N; Divecha, Nullin; D'Santos, C; PROBE Proteomics Platform, Department of Biomedicine, University of Bergen, Bergen, Norway. (2011-02)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 IIα. 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.
Nuclear phosphoinositides and their impact on nuclear functions.Shah, Zahid H; Jones, David R; Sommer, Lilly; Foulger, Rebecca; Bultsma, Yvette; D'Santos, C; Divecha, Nullin; Cancer Research UK Inositide Laboratory, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, M20 4BX, UK. (2013-09-24)Polyphosphoinositides (PPIn) are important lipid molecules whose levels are deregulated in human diseases such as cancer, neurodegenerative and in metabolic syndromes. PPIn are synthesised and degraded by an array of kinases, phosphatases and lipases which are localised to various subcellular compartments and are subject to regulation in response to both extra- and intracellular cues. Changes in the activities of enzymes that metabolise PPIn lead to changes in the profiles of PPIn in different sub-cellular compartments. Understanding how subcellular PPIn are regulated and how they impact on downstream signaling is critical to understand 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 impact on. Over recent years targeted and global proteomic studies have identified a plethora of potential PPIn interacting proteins involved in many aspects of transcription, chromatin remodeling and mRNA maturation suggesting that PPIn signaling within the nucleus represents a largely unexplored novel layer of complexity in the regulation of nuclear functions. This article is protected by copyright. All rights reserved.