PROJECT SUMMARY Inositol is a ubiquitous six-carbon cyclitol that is essential for viability of eukaryotic cells. Inositol compounds play pivotal roles in cell signaling and metabolism, and regulate virtually every aspect of cellular physiology. Consistent with its importance, perturbation of inositol metabolism is associated with pathologies as diverse as neurological and psychiatric illnesses, myopathies, cancer, and diabetes. In this light, it is striking that almost nothing is known about how inositol synthesis is regulated in mammalian cells. The goal of the proposed research is to elucidate the molecular mechanisms that regulate inositol synthesis. To this end, we have exploited yeast genetics to identify novel mechanisms of regulation of inositol synthesis, and mammalian cells to test hypotheses generated by the yeast studies. Using this approach, we identified three novel mechanisms of regulation of inositol synthesis, which are the focus of this proposal. Aim 1 will elucidate the mechanism whereby phosphorylation regulates myo- inositol-3-phosphate synthase (MIPS), which catalyzes the rate-limiting step in inositol de novo synthesis. Aim 2 will test our hypothesis that MIPS is regulated by metabolic channeling of the common intermediate, glucose-6-phosphate, and that down-regulation of inositol synthesis is triggered by increased demand for glycolysis. Aim 3 will determine the physiological significance of transcriptional control of mammalian INO1 expression by inositol pyrophosphate synthase (IP6K1). We expect these studies to generate the first molecular model of regulation of inositol synthesis in mammalian cells. The model will suggest a new paradigm with which to understand how cross talk between glycolysis and inositol synthesis regulates these essential metabolic pathways. The outcome of these studies will contribute to our understanding of essential cellular processes that require inositol, and open the door to the development of new therapeutics for the treatment of bipolar disorder, epilepsy, and other illnesses in which inositol homeostasis is perturbed.