Mechanistic role of phosphatidylinositol 5-phosphate 4-kinase beta in GTP-dependent lysosomal acidification for stress-resilient cell growth and metabolism

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Increased anabolism is a common feature of tumors and several metabolic diseases. The high anabolic state is typically accompanied by systemic suppression of catabolism (e.g., lysosome biogenesis and autophagy). Paradoxically, the anabolic cells increase dependence on the lysosomal degradation pathways to counteract the obligately increased stresses, such as malfunctioned organelle and reactive oxygen species. However, the molecular mechanism of how cells activate lysosomal functions regardless of their anabolic state remains largely unknown. Phosphatidylinositol 5-phosphate 4-kinase (PI5P4K) is a family of enzymes, consisting of PI5P4Kα, β, γ, and converts the lipid second messenger, phosphatidylinositol 5-phosphate (PI5P), to phosphatidylinositol 4,5-phosphate (PI(4,5)P2). The main function of PI5P4K is considered to control PI5P-dependent signaling, as the bulk of PI(4,5)P2 is generated from another family of enzymes, PI4P5Ks. Genetic deletion studies of the three genes in the PI5P4K family (Pip4k2a, Pip4k2b, and Pip4k2c) in mice indicate that PI5P4Kβ plays distinct and critical roles in mediating cellular responses to stress (e.g., nutrient deprivation, ROS) and ultimately affect whole-body insulin sensitivity, growth, obesity, and cancer. Importantly, PI5P4Ks are atypical kinases that have a unique property to use GTP as a phosphodonor. In particular, PI5P4Kβ preferentially uses GTP rather than ATP, and its kinase activity is regulated by physiological GTP concentrations, acting as a cellular GTP sensor for metabolism and tumorigenesis by mechanisms yet to be defined. Pertaining to this proposal, our group has developed isozyme selective PI5P4K inhibitors using newly developed NMR-based screening, and found that treatment of the PI5P4K inhibitors suppressed lysosome acidification. Newly generated GTP-insensitive Pip4k2bF205L/F205L mice developed severe steatosis compared to WT mice, and exhibited increased hypoglycemia upon fasting, resembling the phenotype of autophagy deficiency. We hypothesize that GTP-dependent PI5P4Kβ activation promotes lysosomal acidification to counterbalance the anabolic stress for stress-resilient cellular growth and hepatic functions. Capitalizing on our long-standing, productive collaborations with a number of cutting-edge laboratories, we will define the mechanistic role of PI5P4Kβ in transcriptionally-independent lysosomal acidification and stress-resilient growth. Using the “structural reverse-genetics” framework that we have developed recently, we will dissect and determine the role of kinase activity and scaffolding functions of PI5P4Kβ (Aim 1). We will test the hypothesis that GTP-dependent PI5P4Kβ activity is required for hepatic lysosomal function and whole-body energy homeostasis (Aim 2). Upon completing the proposed research, we will identify the novel stress counteracting system through which GTP-mediated activation of PI5P4Kβ promotes lysosomal activation to support stress-resilient anabolic cell growth and protect mice from the pathogenesis of metabolic diseases.