The Inositol Hexakisphosphate Kinase Family

Abstract

Saiardi et al. (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP 6 ) to diphosphoinositol pentakisphosphate, a "high energy" candidate regulator of cellular trafficking. We have now studied the significance of InsP 6 kinase activity in Saccharomyces cerevisiae by disrupting the kinase gene. These ip6k⌬ cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60 -80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles. Novel activities of the mammalian and yeast InsP 6 kinases were identified; inositol pentakisphosphate (InsP 5 ) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP 4 ), which was further metabolized to a novel compound, tentatively identified as bisdiphosphoinositol trisphosphate. The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase. Kinetic parameters for the mammalian type 1 kinase indicate that InsP 5 (K m ‫؍‬ 1.2 M) and InsP 6 (K m ‫؍‬ 6.7 M) compete for phosphorylation in vivo. This is the first time a PP-InsP 4 synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP 6 . Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged.The very dynamic turnover of the "high energy" diphospho- For example, PP-InsP 5 represents the most potent known inhibitor of AP180-mediated assembly of clathrin cages, a key step in the endocytic retrieval of discharged synaptosomal vesicles (2). Other proteins that participate in intracellular trafficking can bind PP-InsP 5 very tightly, including coatomer (3, 4) and AP2 (5). The high affinity with which PP-InsP 5 binds to myelin proteolipid protein may be important for the vesicular delivery of the latter to the myelin sheath (6).Prior experiments with intact cells have shown that Ins(1,3,4,5,6)P 5 and InsP 6 serve as metabolic stockpiles for the formation of the diphosphorylated inositol polyphosphates (7,8). InsP 6 is the precursor for PP-InsP 5 , which is further phosphorylated to [PP] 2 -InsP 4 (5, 7-10). These reactions appear to take place within a metabolic pool that is separate from that in which Ins(1,3,4,5,6)P 5 and PP-InsP 4 are interconverted (7). Thus, there are two metabolic pools of inositol diphosphates that are turned over in parallel cycles.There are increasing efforts to characterize the activities of the enzymes that regulate the turnover of PP-InsP 4 , PP-InsP 5 , and [PP] 2 -InsP 4 . Several phosphatases (diphosphoinositol polyphosphate phosphohydrolases) that hydrolyze these compounds have been described (11,12). Two forms of InsP 6 kinase (types 1 and 2), derived from distinct genes, have been cloned from mammals (13,14). At least in mammals, the further phosphorylati...

Mammalian inositol polyphosphate multikinase synthesizes inositol 1,4,5-trisphosphate and an inositol pyrophosphate

“…We now report the cloning and characterization of mammalian IPMK (mIPMK). We have discovered an unprecedented substrate specificity whereby mIPMK can synthesize InsP 3 (14). All of the radiolabeled inositol phosphates that we synthesized were purified by HPLC and desalted (15).…”

Telomere maintenance by intracellular signals: New kid on the block?

“…In mammals, such events might involve differential expression of the three isoforms of IP 6 kinase, because each has different affinities for the competing IP 5 and IP 6 substrates (12, 13). Unfortunately, perhaps, the one isoform of IP 6 kinase that is predominantly nuclear (type 2) has the weakest ability to phosphorylate IP 5 to PP-IP 4 (12,13). Furthermore, no one has yet shown that there is an extracellular stimulus that can specifically regulate PP-IP 4 levels, thereby providing opportunities for telomere length to be regulated independent of the many effects that other inositol pyrophosphates can elicit (see above).…”

“…Only one homologue, Kcs1, has been cloned from Saccharomyces cerevisiae (18). All IP6 kinases also phosphorylate IP5 to PP-IP4 (12). Not shown is the hydrolysis of the diester phosphates by specific phosphohydrolases (19).…”

Inositol Hexakisphosphate Kinase 2 Mediates Growth Suppressive and Apoptotic Effects of Interferon-β in Ovarian Carcinoma Cells

“…IP6 is not the only substrate for IP6K1 and IP6K2. Both IP6Ks can also phosphorylate IP5 to yield PP-IP4 (32). Because PEI-TLC could not clearly resolve PP-IP5 and PP-IP4, it is possible the latter compound may also contribute to growth inhibition and apoptosis.…”

“…Inositol PP include PP-IP5 (7 phosphates/inositol), bis-PP-IP4 (8 phosphates/inositol) (31), PP-IP4 (6 phosphates/inositol), and bis-PP-IP3 (7 phosphates/inositol) (32). The specific functional roles of PP-IP5, bis-PP-IP4, and their precursor, IP6, are not clearly understood.…”