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 Pi homeostasis are critical for all life. In Saccharomyces cerevisiae, extracellular [P-1] is sensed by the inositol-hexakisphosphate kinase (IP6K) that synthesizes the intracellular inositol pyrophosphate 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP(7)) as follows: during a period of Pi starvation, there is a decline in cellular [ATP]; the unusually low affinity of IP6Ks for ATP compels 5-InsP(7) levels to fall in parallel (Azevedo, C.,and Saiardi, A. (2017) Trends. Biochem. Sci. 42, 219-231. Hitherto, such P-1 sensing has not been documented in metazoans. Here, using a human intestinal epithelial cell line (HCT116), we show that levels of both 5-InsP(7) and ATP decrease upon [P-1] starvation and subsequently recover during Pi replenishment. However, a separate inositol pyrophosphate, 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP(8)), reacts more dramatically (i.e. with a wider dynamic range and greater sensitivity). To understand this novel InsP(8) response, we characterized kinetic properties of the bifunctional 5-InsP(7) kinase/InsP(8) 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 (InsP(8) in this case) to fluctuate independently of varying precursor (i.e. 5-InsP(7)) pool size. Moreover, we report that InsP(8) phosphatase activities of PPIP5Ks are strongly inhibited by P-1 (40-90% within the 0-1 mM range). For PPIP5K2, P-1 sensing by InsP(8) is amplified by a 2-fold activation of 5-InsP(7) kinase activity by P-1 within the 0-5 mM range. Overall, our data reveal mechanisms that can contribute to specificity in inositol pyrophosphate sig-naling, regulating InsP(8) turnover independently of 5-InsP(7), in response to fluctuations in extracellular supply of a key nutrient.