An evolutionary proteomics approach for the identification of substrates of the cAMP-dependent protein kinase in Saccharomyces cerevisiae

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 thesis, I will discuss my recent work with one of these potential targets, Atg1p.; Atg1p is a key regulator of autophagy, a membrane trafficking pathway that is responsible for much of the protein and organelle turnover occurring during periods of nutrient deprivation. This catabolic pathway is highly conserved and Atg1p homologs exist in essentially all eukaryotic organisms. We have found that the S. cerevisiae Atg1p contains two PKA consensus sites that have been conserved in all budding yeast and C. albicans . My work has shown that Ras/PKA signaling activity does indeed control autophagy in vivo and that Atg1p is phosphorylated at the two predicted sites by PKA. PKA phosphorylation of Atg1p does not effect its protein kinase activity, but appears instead negatively regulate the association of Atg1p with the pre-autophagosomal structure (PAS). We hypothesize that this regulation might prevent Atg1p from reaching specific substrates that are important for the autophagy process.