A biochemical genomics screen for substrates of Ste20p kinase enables the in silico prediction of novel substrates.
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.
Annan
Robert B
RB
Department of Biochemistry, McGill University, Montreal, Quebec, Canada. robert.annan@mail.mcgill.ca
Lee
Anna Y
AY
Reid
Ian D
ID
Sayad
Azin
A
Whiteway
Malcolm
M
Hallett
Michael
M
Thomas
David Y
DY
D000595:Amino Acid Sequence; D016000:Cluster Analysis; D053263:Gene Regulatory Networks / Q000235:genetics; D023281:Genomics / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D047908:Intracellular Signaling Peptides and Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; C078754:STE20 protein, S cerevisiae