Modification and trafficking of RAS and other small GTPases
Small GTPases such as RAS are targeted to membranes by virtue of a series of post-translational modifications of their C-termini. These include prenylation, proteolysis and carboxylmethylation of a ubiquitous CaaX motif and palmitoylation of adjacent cysteines. Because palmitoylation is reversible and regulates subcellular localization we are working on methods to measure endogenous palmitoylation that will allow us to study the regulation of this modification. In collaboration with Shafi Kuchay (U Illinois Chicago) and Michele Pagano (NYU) we recently reported a fourth protein prenyltransferase, GGTase 3, and are interested characterizing its role in resistance to farnesyltransferase inhibitors. In an effort to identify genes involved in RAS trafficking we developed an assay that reports membrane association of KRAS4B and used it in genome-wide screens (in collaboration with David Calderwood, Ben Turk and Joseph Schlessinger at Yale) that identified a number of kinases and SAFB, a nuclear DNA/RNA binding protein that regulates prenyltransferase expression. We are currently working on the mechanisms of action of these hits. Having shown that phosphorylation of KRAS4B on serine 181 weakens its affinity for the plasma membrane we have developed a genetically engineered mouse model (GEMM) to test the significance of this modification in vivo.
Gel filtration of endogenous cytosolic RAS
