The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis

Proteins responsible for P_i homeostasis are critical for all life. In Saccharomyces cerevisiae, extracellular [P_i] is "sensed" by the inositol-hexakisphosphate kinase (IP6K) that synthesizes the intracellular inositol pyrophosphate 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP₇) as follows: during a period of P_i starvation, there is a decline in cellular [ATP]; the unusually low affinity of IP6Ks for ATP compels 5-InsP₇ levels to fall in parallel (Azevedo, C., and Saiardi, A. (2017) Trends. Biochem. Sci. 42, 219-231. Hitherto, such P_i sensing has not been documented in metazoans. Here, using a human intestinal epithelial cell line (HCT116), we show that levels of both 5-InsP₇ and ATP decrease upon [P_i] starvation and subsequently recover during P_i replenishment. However, a separate inositol pyrophosphate, 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP₈), reacts more dramatically (i.e. with a wider dynamic range and greater sensitivity). To understand this novel InsP₈ response, we characterized kinetic properties of the bifunctional 5-InsP₇ kinase/InsP₈ phosphatase activities of full-length diphosphoinositol pentakisphosphate kinases (PPIP5Ks). These data fulfil previously published criteria for any bifunctional kinase/phosphatase to exhibit concentration robustness, permitting levels of the kinase product (InsP₈ in this case) to fluctuate independently of varying precursor (i.e. 5-InsP₇) pool size. Moreover, we report that InsP₈ phosphatase activities of PPIP5Ks are strongly inhibited by P_i (40-90% within the 0-1 mm range). For PPIP5K2, P_i sensing by InsP₈ is amplified by a 2-fold activation of 5-InsP₇ kinase activity by P_i within the 0-5 mm range. Overall, our data reveal mechanisms that can contribute to specificity in inositol pyrophosphate signaling, regulating InsP₈ turnover independently of 5-InsP₇, in response to fluctuations in extracellular supply of a key nutrient.