Abstract
The Ste20/PAK family is involved in many cellular processes, including the regulation of actin-based cytoskeletal dynamics and the activation of MAPK signaling pathways. Despite its numerous roles, few of its substrates have been identified. To better characterize the roles of the yeast Ste20p kinase, we developed an in vitro biochemical genomics screen to identify its substrates. When applied to 539 purified yeast proteins, the screen reported 14 targets of Ste20p phosphorylation. We used the data resulting from our screen to build an in silico predictor to identify Ste20p substrates on a proteome-wide basis. Since kinase-substrate specificity is often mediated by additional binding events at sites distal to the phosphorylation site, the predictor uses the presence/absence of multiple sequence motifs to evaluate potential substrates. Statistical validation estimates a threefold improvement in substrate recovery over random predictions, despite the lack of a single dominant motif that can characterize Ste20p phosphorylation. The set of predicted substrates significantly overrepresents elements of the genetic and physical interaction networks surrounding Ste20p, suggesting that some of the predicted substrates are in vivo targets. We validated this combined experimental and computational approach for identifying kinase substrates by confirming the in vitro phosphorylation of polarisome components Bni1p and Bud6p, thus suggesting a mechanism by which Ste20p effects polarized growth.
Highlights
Protein phosphorylation is a central post-translational modification in signal transduction; underscoring its importance is the observation that roughly 2% of eukaryotic genes encode kinases, and roughly one-third of all intracellular proteins may be phosphorylated on at least one residue [1,2,3]
The combined pools of purified proteins bound to beads were incubated in each of two solutions: a solution containing the kinase domain of Ste20p, expressed and purified from E. coli, with necessary cofactors and c-[P32]-ATP, and a control solution lacking Ste20p kinase
We developed a strategy to aid the discovery of substrates for any given kinase and demonstrated its utility with the yeast Ste20p kinase
Summary
Protein phosphorylation is a central post-translational modification in signal transduction; underscoring its importance is the observation that roughly 2% of eukaryotic genes encode kinases, and roughly one-third of all intracellular proteins may be phosphorylated on at least one residue [1,2,3]. Given the large number of possible substrates for each of the many protein kinases, it is not surprising that the identification of kinase-substrate relationships remains a daunting challenge. Our knowledge of kinase-substrate relationships has expanded using approaches that detect in vitro phosphorylation or in vivo phosphoproteins. In vivo methods include the use of mass spectrometry to generate large-scale profiles of cellular phosphoproteins (reviewed in [10,11]). Recent studies have combined these approaches with the examination of evolutionary conservation and interaction networks to better understand kinasesubstrate relationships [12].While such approaches have expanded our knowledge of kinase-substrate relationships, it is clear that many remain unidentified or uncharacterized by current methods
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