Inositol Pyrophosphate Synthesis by Diphosphoinositol Pentakisphosphate Kinase-1 is Regulated by...

5-diphosphoinositol tetrakisphosphate (5-InsP₇) and bisdiphosphoinositol tetrakisphosphate (InsP₈) are 'energetic' inositol pyrophosphate signaling molecules that regulate bioenergetic homeostasis. Inositol pyrophosphate levels are regulated by diphosphoinositol pentakisphosphate kinases (PPIP5Ks); these are large modular proteins that host a kinase domain (which phosphorylates 5-InsP₇ to InsP₈), a phosphatase domain that catalyzes the reverse reaction, and a polyphosphoinositide-binding domain (PBD). Here, we describe new interactions between these three domains in the context of full-length human PPIP5K1. We determine that InsP₇ kinase activity is dominant when PPIP5K1 is expressed in intact cells; in contrast, we found that InsP₈ phosphatase activity prevails when the enzyme is isolated from its cellular environment. We approach a reconciliation of this disparity by showing that cellular InsP₈ phosphatase activity is inhibited by C₈-PtdIns(4,5)P₂ (IC₅₀ approx. 40 ìM). We recapitulate this phosphatase inhibition with natural PtdIns(4,5)P₂ that was incorporated into large unilamellar vesicles. Additionally, PtdIns(4,5)P₂ increases net InsP₇ kinase activity 5-fold. We oftlinedemonstrate that PtdIns(4,5)P₂ is not itself a phosphatase substrate; its inhibition of InsP₈ phosphatase activity results from an unusual, functional overlap between the phosphatase domain and the PBD. Finally, we discuss the significance of PtdIns(4,5)P₂ as a novel regulator of PPIP5K1, in relation to compartmentalization of InsP₇/InsP₈ signaling in vivo.