Functional characterization of a novel evolutionarily conserved inositol hexakisphosphate kinase in mammalian cells

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Myo-inositol derived signaling molecules come as two different classes of chemical compounds: membrane-resident phosphoinositides and cytosolic inositol polyphosphates. Inositol polyphosphates represent a multifaceted family of signaling molecules comprising the famous calcium release factor Ins(1,4,5)P3 as well as molecules containing a diphospho- moiety known as inositol pyrophosphates. Inositol pyrophosphates control several crucial cellular and physiological processes including chromatin remodeling, apoptosis, mRNA export, and vesicle trafficking. Recent studies have proposed that many of these functions could be attributed to the ability of inositol pyrophosphates to regulate basic metabolism through phosphate homeostasis. The best characterized inositol pyrophosphates are InsP7 and InsP8. In yeast, amoeba and metazoan, the biosynthesis of InsP7 and InsP8 is mediated by two distinct classes of enzymes, Kcs1/IP6K-type and Vip1/PPIP5K-type proteins, respectively. In plants, InsP7 and InsP8 are also present, however no direct homologs of Kcs1/IP6K-type kinases are encoded by plant genomes. Recently, we have identified a new type of InsP6 kinases in plants that are not sequence-related to Kcs1/IP6K-type kinases and can mediate the biosynthesis of InsP7. Interestingly, this novel type of InsP6 kinase is also encoded by the human genome despite the presence of three Kcs1/IP6K-type kinases. The aim of the proposed research is to functionally characterize this novel InsP6 kinase in mammalian cells by employing a series of comprehensive biochemical, biophysical and cell biological techniques. In short, I will explore the inositol polyphosphate kinase activities of this novel enzyme. I will investigate the molecular structure of the InsP6 kinase reaction products using established biochemical and biophysical techniques. Moreover, I will generate knockout mammalian cells of this kinase using CRISPR and will perform metabolic experiments designed to investigate the inositol polyphosphate homeostasis in the loss-of-function cells. Furthermore, I will combine genetic, cell-biological and metabolic experiments to determine the physiological consequences of these kinase deficient cells under several stresses including phosphate starvation. Taken together, the proposed research will help to understand in depth the molecular mechanisms through which inositol pyrophosphates orchestrate several critical cellular processes and to which degree these processes are associated with phosphate homeostasis.