Small GTPases are molecular switches that control a wide variety of cellular processes, including those that regulate cell motility, polarity, vesicular traffic, adhesion, proliferation, differentiation and survival. As such, this superfamily of signaling molecules plays a role in virtually every aspect of cellular physiology and is central to the pathophysiology of disorders as diverse as autoimmunity and cancer. A GTP/GDP cycle regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) constitutes the molecular switch. When in the GTP-bound,“on” conformation, small GTPases associate with and activate a wide variety of effectors. Much has been learned over the past quarter century regarding the biochemical regulation of small GTPases and the pathways controlled by these molecules. More recently, the post-translational modifications, trafficking and membrane targeting of these proteins have come into focus. In our laboratory the overarching theme is the study of how the trafficking of small GTPases relates to their function. Our hypothesis is that the complexity and regulation of the subcellular localization allows for a broader repertoire of functional output.
Ras is not only the prototypical small GTPase but also the prototypical CAAX protein. CAAX proteins are a large family of proteins that are targeted to cellular membranes by virtue of a series of posttranslational modifications that include polyisoprenylation, endoproteolytic cleavage and carboxyl methylation. Ras and related GTPases require membrane targeting for function. Ras is also the oncogene most often mutated in human cancer. Accordingly, the Ras trafficking pathway has been considered an excellent target for anti-cancer drug discovery, a realization that led to the development of farnesyl transferase inhibitors. Among our accomplishments is the molecular cloning and characterization of Icmt, the enzyme that methylates Ras and other CAAX proteins. We are currently performing a structure-function analysis of Icmt and are using a molecular genetic approach to assess the requirement for this enzyme in oncogenesis and tumor progression.
We discovered that post-prenylation processing of CAAX proteins takes place on the cytoplasmic face of the endoplasmic reticulum (ER) and Golgi apparatus and that nascent Ras transits the endomembrane en route to the plasma membrane (PM). This led us to test the hypothesis that intracellular Ras is capable of signaling. To accomplish this we developed a genetically encoded fluorescent probe that reports when and where Ras is activated in living cells. Using this probe we discovered that mitogens stimulate GTP/GDP exchange on the Golgi as well as the PM. We mapped the pathway of Ras activation on the Golgi and discovered that it is regulated by PLC via the Ras GEF RasGRP1. Because RasGRP1 is highly expressed in lymphocytes we have turned our attention to these cells and found that the predominant site of Ras activation is on the Golgi apparatus. Interestingly, RasGRP1 deficiency in mice leads to autoimmunity. Current efforts in the lab include studies designed to elucidate the biological consequences of Ras signaling from various subcellular compartments in both lymphoid and non-lymphoid cells.
Mammalian genomes include three ras genes that encode four protein isoforms. Of these, K-Ras is by far the one most often associated with human cancer. Accordingly, biology specific to K-Ras is of particular interest to cancer biologists. We have recently discovered that the localization of K-Ras on the PM is regulated by a farnesyl-electrostatic switch that is, in turn, regulated by phosphorylation of K-Ras on serine 181 by PKC. Surprisingly, phosphorylated K-Ras discharged from the PM accumulates on intracellular membranes including the outer mitochondrial membrane. Even more surprising, phosphorylated K-Ras promotes apoptosis in a Bcl-XL dependent fashion. This discovery suggests a strategy for treating K-Ras dependent tumors with PKC agonist. We have validated this idea by showing that K-Ras dependent tumors in nude mice are sensitive to the PKC agonist bryostatin-1 but not when serine 181 is mutated to alanine. Studies are now underway to better understand the apoptotic pathway engaged by phospho-K-Ras and to screen for compounds that promote K-Ras phosphorylation and translocation and might thereby be useful as anti-cancer agents.