The inositol pyrophosphate metabolic pathway One of these inositol pyrophosphates, PP-InsP 5, has been shown to directly phosphorylate-specific target proteins in a kinase-independent manner ( 23). These molecules have been reported to regulate vesicle trafficking ( 11), transcription ( 12), chemotaxis ( 13), telomere maintenance ( 14, 15), apoptosis ( 16, 17), and DNA repair ( 18) and to mediate environmental stress responses ( 19- 22). In the early 1990s, two groups working independently discovered a novel subgroup of the inositol phosphate signaling family in which diphosphate groups are added to Ins(1,3,4,5,6)P 5 and InsP 6 ( 8, 9), forming compounds that are now generally described as “inositol pyrophosphates” or “diphosphoinositol polyphosphates” (see Fig. Many additional biological activities have been attributed to these and other inositol phosphates ( 5- 7). An inositol ring with six phosphates (InsP 6) enhances the activity of Dbp5, a key component of a molecular ratchet that winches mRNA out of the nucleus ( 4). The 3,4,5,6-tetrakisphosphate of inositol (Ins(3,4,5,6)P 4) inhibits Cl - channel conductance ( 3). To list just a few examples, a 1,4,5-trisphosphate configuration yields a molecule (Ins(1,4,5)P 3) 2 that gates intracellular Ca 2+ channels ( 2).
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). PPIP5Ks are likely to be important signaling enzymes. Immunopurification of over expressed 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. Analysis by confocal immunofluorescence showed PPIP5K1 to be distributed throughout the cytoplasm but excluded from the nucleus. PPIP5K1 and PPIP5K2 were more active against PP-InsP 5 than InsP 6, both in vitro and in vivo. Overexpression in human embryonic kidney cells of either PPIP5K1 or PPIP5K2 substantially increased levels of (PP) 2-InsP 4, whereas overexpression of a catalytically dead PPIP5K1 D332A mutant had no effect. Recombinant human PPIP5K1, expressed in Escherichia coli, was found to phosphorylate diphosphoinositol pentakisphosphate (PP-InsP 5) to (PP) 2-InsP 4 (V max = 8.3 nmol/mg of protein/min K m = 0.34 μ m). BLAST searches uncovered two human isoforms (PPIP5K1 (160 kDa) and PPIP5K2 (138 kDa)).
Partial amino acid sequence, obtained by mass spectrometry, matched the sequence of a 160-kDa rat protein containing a putative ATP-grasp kinase domain. Here, we describe the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIP5K) that synthesizes (PP) 2-InsP 4.
Recently, osmotic stress was found to increase synthesis of bisdiphosphoinositol tetrakisphosphate ((PP) 2-InsP 4), a high energy inositol pyrophosphate (Pesesse, X., Choi, K., Zhang, T., and Shears, S. 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.
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