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) 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.
Choi
Jae H
JH
Inositide Signaling Group Laboratory of Signal Transduction, NIEHS, National Institutes of Health, DHHS, North Carolina 27709, USA.
Williams
Jason
J
Cho
Jaiesoon
J
Falck
J R
JR
Shears
Stephen B
SB
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D003600:Cytosol / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009997:Osmotic Pressure; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
D007295:Inositol Phosphates; D007527:Isoenzymes; D011994:Recombinant Proteins; C079849:1-diphosphoinositol pentakisphosphate; D017856:Phosphotransferases (Phosphate Group Acceptor); C407349:diphosphoinositol pentakisphosphate kinase
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