Abstract
Mammalian cells utilize multiple signaling mechanisms to protect against the osmotic stress that accompanies plasma membrane ion transport, solute uptake, and turnover of protein and carbohydrates (Schliess, F., and Haussinger, D. (2002) Biol. Chem. 383, 577-583). Recently, osmotic stress was found to increase synthesis of bisdiphosphoinositol tetrakisphosphate ((PP)2-InsP4), a high energy inositol pyrophosphate (Pesesse, X., Choi, K., Zhang, T., and Shears, S. B. (2004) J. Biol. Chem. 279, 43378-43381). Here, we describe the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIP5K) that synthesizes (PP)2-InsP4. Partial amino acid sequence, obtained by mass spectrometry, matched the sequence of a 160-kDa rat protein containing a putative ATP-grasp kinase domain. BLAST searches uncovered two human isoforms (PPIP5K1 (160 kDa) and PPIP5K2 (138 kDa)). Recombinant human PPIP5K1, expressed in Escherichia coli, was found to phosphorylate diphosphoinositol pentakisphosphate (PP-InsP5) to (PP)2-InsP4 (Vmax = 8.3 nmol/mg of protein/min; Km = 0.34 microM). Overexpression in human embryonic kidney cells of either PPIP5K1 or PPIP5K2 substantially increased levels of (PP)2-InsP4, whereas overexpression of a catalytically dead PPIP5K1(D332A) mutant had no effect. PPIP5K1 and PPIP5K2 were more active against PP-InsP5 than InsP6, both in vitro and in vivo. Analysis by confocal immunofluorescence showed PPIP5K1 to be distributed throughout the cytoplasm but excluded from the nucleus. Immunopurification of overexpressed PPIP5K1 from osmotically stressed HEK cells (0.2 M sorbitol; 30 min) revealed a persistent, 3.9 +/- 0.4-fold activation when compared with control cells. PPIP5Ks are likely to be important signaling enzymes.
Highlights
The phosphorylated inositol moiety is viewed as a fundamental signaling entity that the cell utilizes to generate combinatorially complex arrays of communication pathways with multiple functions [1]
We demonstrate here that mammalian PPIP5K1 is a homologue of Vip1 that functions as an PPInsP5 to (PP)-InsP5 kinase both in vitro and in intact cells
Inositol Phosphate Kinase Assays—PP-InsP5 kinase activity during its purification was assayed at 37 °C in 100 l of buffer containing 50 mM KCl, 20 mM HEPES, 20 mM phosphocreatine, 10 mM NaF, 10 mM ATP, 12 mM MgSO4, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml bovine serum albumin, and 0.01 mg/ml phosphocreatine kinase, plus 10,000 dpm of PP-[3H]InsP5; the latter was generated in situ by preincubation for 60 min with [3H]InsP6 plus InsP6 kinase (murine type 1 [35])
Summary
Particular interest as a signaling entity because, alone among the inositol pyrophosphates, its levels are acutely regulated, for example, by thermal stress [19], osmotic shock [21], and cAMP (in a cAMP-dependent protein kinase-independent manner) [32]. During the final stages of this study, York and colleagues [33] described the characterization of a novel InsP6 kinase from Saccharomyces cerevisiae, Vip. The 4/6-PP-InsP5 that is produced from InsP6 by Vip regulates the transcriptional activity of a cyclin/cyclin-dependent kinase complex that controls cellular phosphate accumulation [34]. We demonstrate here that mammalian PPIP5K1 is a homologue of Vip that functions as an PP-InsP5 kinase both in vitro and in intact cells. We show that this enzyme is acutely activated by osmotic stress. We conclude that PPIP5Ks are important enzymes in signal transduction
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