Abstract 27: Understanding mechanisms of nutrient homeostasis: Amino acid availability and regulation of V-ATPase activity

Abstract

Abstract Cellular homeostasis requires accurate sensing of nutrient levels and subsequent regulation of biosynthetic and catabolic cellular processes. In cancer cells, metabolism is frequently disregulated to meet the increased demand for cellular building blocks to enable growth and proliferation. Thorough understanding of how homeostatic processes are regulated is necessary to determine how these processes could be manipulated to stop tumor growth. This study aims to understand amino acid homeostasis, with particular focus on the regulation of lysosomal activity, which allows protein breakdown and release of free amino acids. We have identified a novel mechanism of amino acid homeostasis whereby amino acid starvation leads to increased activity of the Vacuolar-ATPase (V-ATPase), as indicated by measurement of V-ATPase-dependent FITC-dextran fluorescence quenching in isolated lysosomes. The V-ATPase is the proton pump responsible for acidifying intracellular compartments, which is necessary for lysosomal function and protein degradation. This increased activity is due to an increase in the amount of fully assembled holoenzymes, as detected by cell fractionation and Western blotting to assess peripheral subunit association with membranes. Assembly of V-ATPase holoenzymes is a tightly controlled process. We describe the function of these changes with respect to lysosomal protein turnover and mTORC1 activation, in which the V-ATPase plays a critical role. We have also sought to characterize the signaling events that transmit the amino acid starvation signal and controls V-ATPase activity in response to changes in amino acid availability in normal and cancer cells that rely on lysosomal function. These changes do not depend on PI3K or mTORC1 activity, both of which have been linked to changes in V-ATPase activity and assembly in other response to other stimuli, such as glucose and growth factor stimulation and are highly activated across cancers. Together, these results shed light on a poorly understood aspect of cellular biology and novel mechanisms of amino acid homeostasis. Citation Format: Laura A. Stransky, Michael Forgac. Understanding mechanisms of nutrient homeostasis: Amino acid availability and regulation of V-ATPase activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 27.