Inositol pyrophosphate synthesis by diphosphoinositol pentakisphosphate kinase-1 is regulated by phosphatidylinositol(4,5)bisphosphate

5-diphosphoinositol tetrakisphosphate (5-InsP 7 ) and bisdiphosphoinositol tetrakisphosphate (InsP 8 ) 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-InsP 7 to InsP 8 ), 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 InsP 7 kinase activity is dominant when PPIP5K1 is expressed in intact cells; in contrast, we found that InsP 8 phosphatase activity prevails when the enzyme is isolated from its cellular environment. We approach a reconciliation of this disparity by showing that cellular InsP 8 phosphatase activity is inhibited by C 8 -PtdIns(4,5)P 2 (IC 50 approx. 40 ìM). We recapitulate this phosphatase inhibition with natural PtdIns(4,5)P 2 that was incorporated into large unilamellar vesicles. Additionally, PtdIns(4,5)P 2 increases net InsP 7 kinase activity 5-fold. We oftlinedemonstrate that PtdIns(4,5)P 2 is not itself a phosphatase substrate; its inhibition of InsP 8 phosphatase activity results from an unusual, functional overlap between the phosphatase domain and the PBD. Finally, we discuss the significance of PtdIns(4,5)P 2 as a novel regulator of PPIP5K1, in relation to compartmentalization of InsP 7 /InsP 8 signaling in vivo .