The Receptor Tyrosine Kinase/Ras/Raf-MEK-ERK signaling pathway is highly conserved and utilized repeatedly during animal development. Despite being one of the best-studied pathways, the mechanism(s) by which the Raf kinase is activated have not been elucidated. Raf activation appears to be a multi-step process including recruitment to the cell membrane by activated Ras. In addition to Ras, many proteins have been implicated in Raf activation, such as KSR, SUR-8, and CNK. Mutations in Drosophila CNK perturb the Ras pathway, genetic epistasis suggests CNK functions closely with Ras and Raf, and binding studies indicate that CNK can bind Raf. However, what role CNK plays in Ras signaling/Raf activation remains unclear. C. elegans has one CNK homolog R01H10.8, we named
cnk-1. We have found that
cnk-1(RNAi) has no discernible phenotypes in a wild-type background, though has ras-like phenotypes in the
ksr-1 and hypomorphic
lin-45 raf backgrounds. To further address the role of
cnk-1 in C. elegans Ras signaling we collaborated with Agneta Ronnlund and Simon Tuck to obtain a deletion allele,
cnk-1(
sv39). Surprisingly the
cnk-1 deletion mimics the RNAi results. Thus
cnk-1 functions as a nonessential positive regulator of Ras signaling in C. elegans similar to
sur-8 and
ksr-1. We are analyzing the phenotypes of the
cnk-1 deletion in different sensitized genetic backgrounds to gain a better understanding of how it functions in the pathway.
cnk-1 is predicted to encode a protein with multiple protein-protein interaction domains, suggesting that CNK-1 may function as a scaffold or adaptor for Raf and as yet unidentified proteins. Identification of proteins that function closely with CNK-1 may lend insight into how CNK-1 functions to facilitate Ras signaling. One approach we are taking is to screen for CNK-1 interacting proteins in the yeast two-hybrid system. Thus far we have identified one intriguing candidate. We are currently verifying the validity of the interaction and testing the gene by RNAi for a role in Ras signaling. As another approach to identify genes that may function closely with
cnk-1 we are performing a RNAi feeding screen to identify genes that enhance the ras-like lethality of
ksr-1. In a screen of the Chromosome I RNA feeding library we have identified several new
ksr-1 enhancers. Interestingly, the identities of some enhancers suggest a possible role for double-stranded RNA processing in the regulation of the Ras pathway. We are currently testing these
ksr-1 enhancers to determine if they are likely to function closely with
cnk-1 or at another step of the pathway.