Understanding inositol pyrophosphate metabolism and function: Kinetic characterization of the DIPPs

Abstract

We illuminate inositol pyrophosphate turnover and cell-signaling activities, by showing that regulation of yeast cyclin-kinase by 1-InsP7 is not conserved for mammalian CDK5, and by kinetically characterizing Ddp1p/DIPP-mediated dephosphorylation of 1-InsP7, 5-InsP7 and InsP8. Each phosphatase exhibited similar Km values for every substrate (range: 35-148 nM). The rank order of kcat values (1-InsP7 > 5-InsP7 = InsP8) was identical for each enzyme, although DIPP1 was 10-60 fold more active than DIPP2α/β and DIPP3α/β. We demonstrate InsP8 dephosphorylation preferentially progresses through 1-InsP7. Conversely, we conclude that the more metabolically and functionally significant steady-state route of InsP8 synthesis proceeds via 5-InsP7.

A Novel Inositol Pyrophosphate Phosphatase in Saccharomyces cerevisiae

“…IP 6 was purchased from Sigma. Four isoforms of IP 7 (5-diphosphoinositol pentakisphosphate (5PP-IP 5 ), non-hydrolyzable 5-methylene-bisphosphonate inositol pentakisphosphate (5PCP-IP 5 ), 1PP-IP 5 , and non-hydrolyzable 1-methylene-bisphosphonate inositol pentakisphosphate (1PCP-IP 5 )) and IP 8 (3,5-bisdiphosphoinositol tetrakisphosphate (3,5PP-IP 4 )) were synthesized following the methods described previously (13)(14)(15).…”

“…In wild-type yeast, the most abundant isoform, diphosphoinositol pentakisphosphate (PP-IP 5 or IP 7 ), 5 consists of a fully phosphorylated six carbon myo-inositol ring further pyrophosphorylated at one of the carbons. Two physiologically relevant isomers of IP 7 include 1PP-IP 5 and 5PP-IP 5 in which the pyrophosphate group is found at the 1-position or the 5-position, respectively. Sequential phosphorylation of IP 7 results in bisdiphosphoinositol tetrakisphosphate (1,5PP-IP 4 or IP 8 ) that is pyrophosphorylated at both the 1st and 5th positions (1,2).…”

“…The inositol hexakisphosphate kinases synthesize IP 7 , the most abundant inositol pyrophosphate in the cell; they add the ␤-phosphate to IP 6 at the 5th position generating 5PP-IP 5 (2). Yeast possess a single inositol hexakisphosphate kinase gene, KCS1, whereas mammals possess three homologous genes encoding inositol hexakisphosphate kinase (Fig.…”

“…4). IP 8 is synthesized by a PP-IP 5 kinase that adds the ␤-phosphate to IP 7 at the 1-position to make 1,5PP-IP 4 ; this enzyme can also use IP 6 as a substrate, generating 1PP-IP 5 . Mammalian genomes have two * This work was supported by National Science Foundation CAREER Grant MCB-1253809 (to A. C. R.) and a Georgetown University Pilot Grant (to R. J. R.).…”

Inositol pyrophosphates as multifaceted metabolites in the regulation of mammalian signaling networks

“…The de-phosphorylation of inositol pyrophosphates to IP 6 or IP 5 is catalyzed by the enzyme diphosphoinositol polyphosphate phosphohydrolase (DIPP) (Safrany and Shears 1998;Safrany et al 1999;Caffrey et al 2000;Kilari et al 2013) (Figure 1). Compared to 5-IP 7 , 1-IP 7 seems to be a better substrate for DIPP (Lonetti et al 2011;Kilari et al 2013). This bias may be the reason mammalian cells produce higher levels of 5-IP 7 than 1-IP 7 .…”