The binding of GTP activates ras, leading to a structural change in the effector domain, permitting effector protein binding and subsequent downstream signaling. Raf and ralGDS have been identified as mammalian proteins which bind to the activated ras effector domain. The ras interacting regions of raf and ralGDS are small but have no primary sequence homology. This proposal characterizes the interactions between ras and its mammalian effectors. The primary structural requirements for ras -effector interaction will be mapped using codon based random mutagenesis of both the ras interacting domains of ras effector proteins and the ras effector domain. Mutants that permit or block the interaction between ras and its partner proteins will be selected using a yeast two hybrid screen for protein-protein interaction. Differences in the interactions between ras and its effectors will be examined by looking for mutants that are pathway specific. Specific contacts between residues of ras and the ras effectors will be identified by looking for suppressers of ras interaction domain mutants that don't bind to ras. Mutants will be used for the dissection of cellular signaling by ras and will be tested for their biological and biochemical effects. This information is essential for an understanding of the mechanism by which ras acts as a signal transducing protein.
| Winkler, D G; Cutler Jr, R E; Drugan, J K et al. (1998) Identification of residues in the cysteine-rich domain of Raf-1 that control Ras binding and Raf-1 activity. J Biol Chem 273:21578-84 |
| Winkler, D G; Johnson, J C; Cooper, J A et al. (1997) Identification and characterization of mutations in Ha-Ras that selectively decrease binding to cRaf-1. J Biol Chem 272:24402-9 |
