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