A central issue in healthy and diseased cells is how cells sense signals from the outside, and how these signals are used to organize cellular responses. The small GTPase families Ras and Rho are intimately involved in these processes, and mutations in them often lead to the disease state. Thus it has been known for many years that activating mutations in Ras are common in cancer cells, and it is widely believed that Ras mutations are essential for the genesis of mammalian tumors. Most small GTPases carry a CAAX amino acid motif at their C terminus, where C is cysteine, AA are usually two aliphatic amino acids, and X specifies whether the protein will be modified with the 15 carbon farnesyl lipid or the 20 carbon geranylgeranyl lipid. Ras function in the cell requires the CAAX sequence, and for activated Ras to be tumorigenic, the CAAX sequence must be modified by a prenylation pathway. For this reason the pathway is a potential target for anticancer drug therapy. Recent drug development efforts have validated this assumption, with several promising new inhibitors of the pathway now in clinical trials. Much remains to be known about many the prenylation pathway: which of the small G proteins must be methylated for them to be modulated, how methylation levels are controlled by extra- and intracellular signals, how the three partners in the prenylation pathway are regulated if they are, the consequences for cell morphology and response to signals when various of the prenylation steps are reduced or absent, and the role that methylation plays in the very earliest steps of G-coupled response to ligand, are some of the outstanding questions addressed in this proposal.
The aim of this research is to use Dictyostelium as a model system to understand these important questions. Preliminary experiments reveal that deletions in the methylation gene block early signaling and response to cAMP, but not cell growth. Various signaling proteins are mislocalized in this deletion, chemotaxis and cAMP responses are disrupted, and the ability of cells to develop into a coherent tissue is blocked. These observations will be extended using assays that will correlate cellular behavior with molecular changes, and by developing a panel of mutants deficient in each step in the prenylation pathway.
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