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J. Biol. Chem., Vol. 282, Issue 42, 30763-30775, October 19, 2007

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

Jae H. Choi, Jason Williams, Jaiesoon Cho, J. R. Falck^¶, and Stephen B. Shears¹

From the Inositide Signaling Group Laboratory of Signal Transduction and the Protein Microcharacterization Core Facility, Laboratory of Structural Biology, NIEHS, National Institutes of Health, DHHS, Research Triangle Park, North Carolina 27709 and the ^¶Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9038

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 (V_max = 8.3 nmol/mg of protein/min; K_m = 0.34 µM). 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.

Received for publication, June 6, 2007 , and in revised form, July 23, 2007.

^* This work was supported by grants from the Intramural Research Program of the NIEHS/National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: NIEHS, National Institutes of Health, DHHS, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-0793; E-mail: shears{at}niehs.nih.gov.

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