Physiology of Class III PI 3-kinase Signaling 2

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DESCRIPTION (provided by applicant): Autophagy is a cellular response to nutrient stress, in which the formation of a double-walled membrane structure sequesters cytosolic components and organelles and delivers them to the lysosome for degradation. This liberates nutrients for use in new biosynthetic activity. Autophagy is critical for perinatal survival in mice, plays a role in normal tissue maintenance in neurons, hepatocytes, and pancreatic beta cells, and contributes to innate immune responses to pathogens. Its activation under pathological conditions leads to disease states such as muscle wasting, and decreases in autophagic activity may contribute to neurological decline in aging and in neurodegenerative disease. Thus, pharmacological modulation of autophagy may have significant clinical benefit. One approach to modulating autophagy would be through the Class III PI 3-kinase, hVps34, which is required for autophagy in yeast, flies and mammalian cells. hVps34 exists in multi-protein complexes containing components specific for autophagy (Atg14L) or vesicular trafficking (UVRAG), as well as components that function in both these pathways (hVps15, beclin-1). The mechanisms that regulate hVps34 activity, its recruitment to these complexes, and their subcellular localization, are not well understood. This proposal addresses these questions through a focused analysis of hVps34 regulation in mammalian cells and in zebrafish. Aim 1 uses a chemical genetic approach to examine the regulation of hVps34 by the hVps15 protein kinase. It is based on our exciting data showing that the binding of hVps15 to hVps34, previously thought to require hVps15 activity, is in fact kinase independent and only requires hVps15 binding to ATP. Using hVps15 mutants that are able to utilize ATP analogues, we will define kinase dependent and independent signaling by hVps15, and identify hVps15 substrates. Aim 2 examines the dynamics of hVps34 complex formation, both at the whole cell level and, using fluorescence recovery after photobleaching (FRAP), on the autophagosomal membrane. We will determine whether nutrient starvation regulates subunit exchange between hVps34 complexes, and ask whether hVps34-associated proteins are recruited in tandem, or individually, to autophagosomal membranes. Aim 3 uses fluorescence fluctuation spectroscopy, a method able to define the stoichiometry of cytosolic hVps34 complexes in living cells, to measure the regulation of complex formation by nutrients. Finally, Aim 4 uses novel mutants that selectively disrupt hVps34 binding to calmodulin and hVps15 binding to Rab5 and Rab7, block hVps34 degradation, and abolish hVps34 lipid kinase activity while preserving its protein kinase activity. These mutants will be used in a knockdown/rescue approach in cultured cells and in zebrafish, to define mechanisms that regulate hVps34 signaling in vivo. Taken together, these studies will provide important new information on how hVps34 is regulated by nutrient stress, and how it is targeted to autophagosomal membranes. Given that hVps34 is one of the key kinases involved in autophagy, a better understanding of its regulation will lead to new insights into this critical cellular process. PUBLIC HEALTH RELEVANCE: Normal tissues respond to changes in nutrient availability by activating a process known as autophagy, in which cellular contents are degraded to small molecules (amino acids, sugars and fats) that can be used to support continued cell survival. A growing body of data suggests that abnormal regulation of autophagy can lead to human disease, ranging from muscle wasting to neurodegeneration and aging. This proposal studies the regulation hVps34, a key enzyme that is required for autophagy, with the hope that a better understanding of the mechanisms to drive autophagy can be exploited for the development of new pharmaceuticals for the treatment of degenerative diseases.