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.

Rho5p is involved in mediating the osmotic stress response in Saccharomyces cerevisiae, and its activity is regulated via Msi1p and Npr1p by phosphorylation and ubiquitination.

Small GTPases of the Rho family act as molecular switches, and modulation of the GTP-bound state of Rho proteins is a well-characterized means of regulating their signaling activity in vivo. In contrast, the regulation of Rho-type GTPases by posttranslational modifications is poorly understood. Here, we present evidence of the control of the Saccharomyces cerevisiae Rho-type GTPase Rho5p by phosphorylation and ubiquitination. Rho5p binds to Ste50p, and the expression of the activated RHO5(Q91H) allele in an Deltaste50 strain is lethal under conditions of osmotic stress. An overexpression screen identified RGD2 and MSI1 as being high-copy suppressors of the osmotic sensitivity of this lethality. Rgd2p had been identified as being a possible Rho5p GTPase-activating protein based on an in vitro assay; this result supports its function as a regulator of Rho5p activity in vivo. MSI1 was previously identified as being a suppressor of hyperactive Ras/cyclic AMP signaling, where it antagonizes Npr1p kinase activity and promotes ubiquitination. Here, we show that Msi1p also acts via Npr1p to suppress activated Rho5p signaling. Rho5p is ubiquitinated, and its expression is lethal in a strain that is compromised for proteasome activity. These data identify Rho5p as being a target of Msi1p/Npr1p regulation and describe a regulatory circuit involving phosphorylation and ubiquitination.