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

The 5-diphosphoinositol pentakisphosphate (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) ~40 μM). 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 demonstrate 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.