Autophagy is an evolutionarily conserved, degradative pathway that has been implicated in a number of physiological processes such as development and aging as well as cancer and innate immunity. This pathway is important for cell survival in starvation and is considered as a potential target for therapeutic intervention in a number of pathological conditions. Therefore, it is important that we develop a thorough understanding of the mechanisms regulating this trafficking pathway. Autophagy was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during periods of starvation. During autophagy, an isolation membrane emanates from a nucleation site that is known as the phagophore assembly site (PAS). This membrane encapsulates nearby cytoplasm to form an autophagosome that is ultimately targeted to the vacuole/lysosome for degradation. The small molecules produced are then recycled and used by cells during this period of starvation. Autophagy activity is highly regulated and multiple signaling pathways are known to target a complex of proteins that contains the Atg1 protein kinase. Atg1 protein kinase activity is essential for normal autophagy in all eukaryotes and appears to be controlled tightly by a number of kinases, which target this enzyme and its associated protein partners. Our data and that of others have established that Atg1 activity is regulated, at least in part, by protein phosphorylation. In this work, we identified a particular phosphorylation event on Atg1 as an important control point within the autophagy pathway in Saccharomyces cerevisiae. This phosphorylation occurs at a threonine residue, T226, within the Atg1 activation loop that is conserved in all Atg1 orthologs. This activation loop phosphorylation is essential for Atg1 kinase activity and the induction of autophagy. The data also suggested that promoting this autophosphorylation is a primary role for two key conserved regulators of Atg1 activi (open full item for complete abstract)

Eukaryotic cells utilize a network of signal transduction pathways to sense their environment and control their growth and proliferation. Protein kinases are a large group of enzymes that coordinate responses to extracellular and intracellular stimuli via phosphorylation of specific downstream targets. In S. cerevisiae , growth is controlled, in part, by the Ras signaling pathway via the cAMP-dependent protein kinase, PKA. PKA is a serine/threonine-specific protein kinase that has been shown to regulate any aspects of cell growth and metabolism in this budding yeast and other eukaryotes. Unfortunately, finding protein kinase substrates by conventional methods is a difficult and time-consuming task. As a result, few targets of any given protein kinase are known. To simplify this task, we developed an evolutionary proteomics strategy for the identification of PKA substrates in S. cerevisiae and related yeast species. This evolutionary proteomics approach is sequenced-based and takes advantage of the fact that most PKA substrates contain the consensus sequence, R-R-x-S/T-B. In this consensus, “x” refers to any amino acid, “B” to hydrophobic residues and “S” or “T” to the site of phosphorylation. The general approach consists of two basic steps. In the first, we identified all of the proteins in the S. cerevisiae proteome that contain this PKA target consensus sequence. In the second, we asked whether these potential target sites are conserved in the orthologous proteins present in other budding yeast species. For this latter step, we used the recently released genome sequences of six different yeast, including five Saccharomyces species and Candida albicans. The underlying premise of this approach is that PKA sites important for general aspects of cell biology are more likely to be conserved across these evolutionary distances. We are presently testing this basic premise with a small number of proteins predicted to be physiologically relevant PKA substrates. In this th (open full item for complete abstract)