Proteins responsible for Pi homeostasis are critical for all life. In Saccharomyces cerevisiae, extracellular [Pi] 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 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 (Azevedo, C., and Saiardi, A. (2017) Trends. Biochem. Sci. 42, 219-231. 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, 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 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.