Many, probably most, kinase substrates are phosphorylated by more than one protein kinase. This redundancy makes it hard to identify kinase substrates using genetic or biochemical assays.  We reasoned that, in the case of redundant phosphorylation by two protein kinases, if a phosphorylation event is important to the cell, deletion of both of the redundant kinases, would produce a dead or slow-growing cell. 

Therefore we have screened for synthetic genetic interactions between genes encoding protein kinases.  So far, we have identified 30 interactions.  We are now characterizing the double kinase mutants and looking for substrates that are phosphorylated by both of the kinase genes using SDL and other approaches. 

The actin cytoskeleton proteins Rvs167 and Rvs161 are yeast homologs of amphiphysin, a BAR domain protein that has been shown to bind to phospholipids vesicles and promote curvature of membranes in vitro.  Originally we started working on Rvs167 because we found  it was a substrate of the Cdk Pcl-Pho85 and we have gone on to explore its roles in polarizing the actin cytoskeleton, endocytosis, and mating. 

Now we are exploring the role of the BAR domain of Rvs167 and Rvs161.  First we are making mutants that we predict will (and will not) disrupt the membrane binding activity of these BAR domain proteins.  Second, we are putting the mutant Rvs167 and Rvs161 in yeast and assaying localization, protein-protein interactions, response to environmental changes, genetic interactions, and cell biology.   Finally, we will assay the mutated Rvs proteins in biochemical assays for membrane binding and bending.  Our goal is to find out what events and processes in yeast require membrane bending by the Rvs proteins.