The Significance of the Bifunctional Kinase/Phosphatase Activities of PPIP5Ks for Coupling Inositol Pyrophosphate Cell-Signaling to Cellular Phosphate Homeostasis. [Metabolism]

Proteins responsible for Pi homeostasis are critical for all life. In Saccharomyces cerevisiae, extracellular [Pi] is 'sensed' by the IP6K that synthesizes the intracellular inositol pyrophosphate, 5-InsP7, 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-InsP7 levels to fall in parallel (Wild et al., 2016, Science, 352:986). Hitherto, such Pi-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 [Pi] starvation, and subsequently recover during Pi replenishment. However, a separate inositol pyrophosphate, 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 PPIP5Ks. These data fulfill 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 Pi (40-90% within the 0-1 mM range). For PPIP5K2, Pi-sensing by InsP8 is amplified by a 2-fold activation of 5-InsP7 kinase activity by Pi 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.

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