Ras is the oncogene most often associated with human cancer. Accordingly, Ras is an attractive target for anti-cancer drug discovery. Ras proteins are GTPases that are biologically active only when associated with cellular membranes. Ras is the founding member of a large family of proteins that are targeted secondarily to cellular membranes by the posttranslational modification of a C-terminal CAAX motif. CAAX sequences are modified by prenylation, proteolysis and carboxyl methylation, reactions catalyzed respectively by farnesyl or geranyl- geranyltransferases, Ras converting enzyme 1 (Reel) and isoprenylcysteine carboxyl methyltransferase (Icmt). Farnesyl transferase inhibitors (FTIs) have been developed as anti-cancer drugs. Recent evidence that cells deficient in Reel or Icmt are resistant to transformation by Ras has sparked heightened interest in these enzymes as drug targets. Cloned in our laboratory and shown to be an intrinsic ER membrane protein, little is known about the structure, enzymology, regulation and biological function of Icmt. We have generated a library of Icmt mutants and developed methods to study the structure and function of Icmt. A structure/function analysis of Icmt is the subject of this proposal. The Specific Aims are: 1. Biochemical analysis of Icmt. Using point and truncation mutants, photoaffinity and chemical crosslinking of substrates and co-immunoprecipitation we will map catalytic and regulatory domains of Icmt. Using conventional and novel approaches we will map the topology of this multiple membrane spanning enzyme. 2. Functional analysis of Icmt in vitro: role in small GTPase signaling. Using mouse fibroblasts deficient in Icmt and human cells in which the Icmt gene is silenced we will study the role of Icmt in the function and stability of Ras, Ral, Rho and Rab GTPases. We will also study, at the protein level, the expression of Icmt in normal and tumor cells 3. Functional analysis of Icmt in vivo: role in an H-Ras driven mouse mammary tumor model. Using Icmtflox/flox mice we will test the hypothesis that Icmt is required for oncogenesis and tumor maintenance in vivo using a murine model of Ras-driven mammary carcinoma that is both tissue specific and temporally controllable. The studies proposed in this application will elucidate the biochemistry and biology of an enzyme that modifies a host of signaling GTPases and will inform the ongoing effort to develop anti-cancer drugs that act on the Ras trafficking pathway. [unreadable] [unreadable] [unreadable]