Purification, Sequencing, and Molecular Identification of a Mammalian PP-InsP5 Kinase That Is Activated When Cells Are Exposed to Hyperosmotic Stress.

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)]. Biol. Chem. 279, 43378-43381). Here, we describe the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIPSK) 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 ATPgrasp kinase domain. BLAST searches uncovered two human isoforms (PPIP5K1 (160 kDa) and PPIPSK2 (138 kDa)). Recombinant human PPIPSK1, expressed in Escherichia coli, was found to phosphorylate diphosphoinositol pentakisphosphate (PP-lnsP5) to (PP)2-InsP4 (Vmax = 8.3 nmol/mg of protein/min; Km = 0.34 μM). Overexpression in human embryonic kidney cells of either PPIPSK1 or PPIPSK2 substantially increased levels of (PP)2-InsP4, whereas overexpression of a catalytically dead PPIPSK1D332A mutant had no effect. PPIPSK1 and PPIP5K2 were more active against PP-InsP5 than InsP6, both in vitro and in vivo. Analysis by confocal immunofluorescence showed PPIPSK1 to be distributed throughout the cytoplasm but excluded from the nucleus. Immunopurification of overexpressed PPIPSK1 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. PPlPSKs are likely to be important signaling enzymes. [ABSTRACT FROM AUTHOR]