An evolutionary proteomics approach for the identification of PKA targets in Saccharomyces cerevisiae identifies Atg1 and Atg13, two proteins that play a central role in the regulation of autophagy by the Ras/PKA pathway and the Tor pathway

A cell in its natural environment spends most of its time in a quiescent resting state known as G0 in mammals and stationary phase in yeast. A constant challenge the cell faces is then to appropriately determine when the conditions, including nutrient availability, are favorable for it to grow. Since growth involves massive energy expenditure and a vast remodeling of the cell's genetic program, a correct determination very likely means the difference between survival and death. Therefore, this crucial decision is both very tightly regulated and extensively coordinated in time and space. This coordination involves a synthesis between internal factors, and environmental clues such as the availability of essential nutrients and favorable conditions. The cell typically accomplishes this complex task through the combined actions of multiple signaling pathways. These pathways link cells to the extracellular environment by providing nutrient-sensing and noxious stress information and determining the appropriate response to any given situation through the activation of specific cellular programs. In essence, these signaling pathways act as molecular switches that engage and maintain the cell into a growth program when the conditions are favorable. This is done through in part through the downregulation of catabolic processes refractory to growth and the upregulation of anabolic processes conducive to it.;In the budding yeast Saccharomyces cerevisiae, two of the most important signaling pathways that regulate growth in response to the availability of nutrients are the Tor (Target Of Rapamycin) kinase pathway and the Ras/cAMP dependent protein kinase (PKA) pathway. Inhibition of either of these pathways leads to a growth arrest similar to that induced by nutrient starvation, and the cell typically enters into a resting, stationary phase that allows it to survive for prolonged periods of time in adverse conditions. In order to understand how the Tor and PKA kinases regulate growth, it is essential to identify all the substrates of these enzymes. However, the identification of protein kinase targets has proven to be an extremely difficult task.;In Chapter 2, we describe an evolutionary proteomics approach for the identification of PKA targets in the budding yeast, S. Cerevisiae . In this method, we use a previously determined PKA target consensus site and mine the yeast proteome for all occurrences of this consensus. Next, we ask if these sites have been conserved through evolution in related yeast species. We found that evolutionary conservation of a PKA site very strongly correlated with the likelihood of that site being recognized by PKA in an in vitro phosphorylation assay. This approach was successful in identifying 44 novel substrates of PKA in yeast. One particularly interesting subset of targets, the AuTophaGy-related proteins Atg1, Atg13 and Atg18, was involved in the catabolic process of autophagy. Autophagy has been implicated in a number of cellular processes including aging, development and cancer. In yeast, this degradative process is fully induced following nutrient limitation and allows the cell to survive prolonged periods of starvation. Previous work in yeast has shown that both the Tor and the Ras/pathways negatively regulate autophagy during the normal, logarithmic (log) phase of growth. However, the precise mechanism of this inhibition is not clear. In the last part of this chapter, we focus on one particular PKA target, Atg1. We show that Atg1 is phosphorylated by PKA, and that this phosphorylation inhibits autophagy in part through the regulation of Atg1 localization.;In chapter 3, we extend our analysis to Atg13. We find that this protein is a critical target of the Ras/PKA pathway. Atg13 is also regulated by the Tor pathway, and our data suggest that it might be a nexus of signal integration within the autophagy machinery. Tor and PKA appear to respond to different nutritional cues to provide separate inputs in the regulation of Atg13, thus regulating different aspects of the autophagy process.