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Abstract: Genetic manipulation of diphosphoinositol polyphosphate synthesis impacts many biological processes (reviewed in S.B. Shears, Biochem. J. 377, 2004, 265–280). These observations lacked a cell-signalling context, until the recent discovery that bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4 or “InsP8”) accumulates rapidly in mammalian cells in response to hyperosmotic stress (X. Pesesse, K. Choi, T. Zhang, and S. B. Shears J. Biol. Chem. 279, 2004, 43378–43381). We now investigate how widely applicable is this new stress-response. [PP]2-InsP4 did not respond to mechanical strain or oxidative stress in mammalian cells. Furthermore, despite tight conservation of many molecular stress responses across the phylogenetic spectrum, we show that cellular [PP]2-InsP4 levels do not respond significantly to osmotic imbalance, heat stress and salt toxicity in Saccharomyces cerevisiae. In contrast, we show that [PP]2-InsP4 is a novel sensor of mild thermal stress in mammalian cells: [PP]2-InsP4 levels increased 3–4 fold when cells were cooled from 37 to 33 °C, or heated to 42 °C. Increases in [PP]2-InsP4 levels following heat-shock were evident <5 min, and reversible (t 1/2 =7 min) once cells were returned to 37 °C. These responses were blocked by pharmacological inhibition of the ERK/MEK pathway. Additional control processes may lie upstream of [PP]2-InsP4 synthesis, which was synergistically activated when heat stress and osmotic stress were combined. Our data add to the repertoire of signaling responses following thermal challenges, a topic of current interest for its possible therapeutic value. [Copyright &y& Elsevier]

Abstract: To understand how a signaling molecule''s activities are regulated, we need insight into the processes controlling the dynamic balance between its synthesis and degradation. For the Ins(1,3,4,5,6)P5 signal, this information is woefully inadequate. For example, the only known cytosolic enzyme with the capacity to degrade Ins(1,3,4,5,6)P5 is the tumour-suppressor PTEN [J.J. Caffrey, T. Darden, M.R. Wenk, S.B. Shears, FEBS Lett. 499 (2001) 6 ], but the biological relevance has been questioned by others [E.A. Orchiston, D. Bennett, N.R. Leslie, R.G. Clarke, L. Winward, C.P. Downes, S.T. Safrany, J. Biol. Chem. 279 (2004) 1116 ]. The current study emphasizes the role of physiological levels of PTEN in Ins(1,3,4,5,6)P5 homeostasis. We employed two cell models. First, we used a human U87MG glioblastoma PTEN-null cell line that hosts an ecdysone-inducible PTEN expression system. Second, the human H1299 bronchial cell line, in which PTEN is hypomorphic due to promoter methylation, has been stably transfected with physiologically relevant levels of PTEN. In both models, a novel consequence of PTEN expression was to increase Ins(1,3,4,5,6)P5 pool size by 30–40% (p <0.01); this response was wortmannin-insensitive and, therefore, independent of the PtdIns 3-kinase pathway. In U87MG cells, induction of the G129R catalytically inactive PTEN mutant did not affect Ins(1,3,4,5,6)P5 levels. PTEN induction did not alter the expression of enzymes participating in Ins(1,3,4,5,6)P5 synthesis. Another effect of PTEN expression in U87MG cells was to decrease InsP6 levels by 13% (p <0.02). The InsP6-phosphatase, MIPP, may be responsible for the latter effect; we show that recombinant human MIPP dephosphorylates InsP6 to d/l-Ins(1,2,4,5,6)P5, levels of which increased 60% (p <0.05) following PTEN expression in U87MG cells. Overall, our data add higher inositol phosphates to the list of important cellular regulators [Y. Huang, R.P. Wernyj, D.D. Norton, P. Precht, M.C. Seminario, R.L. Wange, Oncogene, 24 (2005) 3819 ] the levels of which are modulated by expression of the highly pleiotropic PTEN protein. [Copyright &y& Elsevier]

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP 7 , is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP 7 regulation in β-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP 7 . Although the inhibitors have diverse targets, they all perturbed IP 7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP 7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high K m for ATP makes IP 7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP 7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP 7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic β-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP 7 's role in cellular signal transduction is likely to have been underestimated., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)