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

Highlights

• Understanding inositol pyrophosphate turnover through kinetic study of Ddp1p/DIPPs.

• k_cat values for 1-InsP₇ are 5–20-fold higher than those for 5-InsP₇ and InsP_8.

• InsP₇ does not regulate CDK5, the human homologue of the yeast Pho85 cyclin kinase.

• Kinetics imply differential expression of DIPP isoforms sets signaling sensitivity.

• Metabolically and functionally separate routes for InsP₈ synthesis and hydrolysis.

Abstract

We illuminate the metabolism and the cell-signaling activities of inositol pyrophosphates, 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 K_m values for every substrate (range: 35–148 nM). The rank order of k_cat values (1-InsP₇ > 5-InsP₇ = InsP₈) was identical for each enzyme, although DIPP1 was 10- to 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.