Caenorhabditis elegans CNK-1 promotes Raf activation but is not essential for Ras/Raf signaling

Abstract

Connector enhancer of Ksr (CNK) is a conserved multidomain protein essential for Ras signaling in Drosophila melanogaster and thought to be involved in Raf kinase activation. However, the precise role of CNK in Ras signaling is not known, and mammalian CNKs are proposed to have distinct functions. Caenorhabditis elegans has a single CNK homologue, cnk-1. Here, we describe the role of cnk-1 in C. elegans Ras signaling and its requirements for LIN-45 Raf activation. We find that cnk-1 positively regulates multiple Ras signaling events during development, but, unlike Drosophila CNK, cnk-1 does not appear to be essential for signaling. cnk-1 mutants appear to be normal but show cell-type-specific genetic interactions with mutations in two other Ras pathway scaffolds͞adaptors ksr-1 and sur-8. Genetic epistasis using various activated LIN-45 Raf transgenes shows that CNK-1 promotes LIN-45 Raf activation at a step between the dephosphorylation of inhibitory sites in the regulatory domain and activating phosphorylation in the kinase domain. Our data are consistent with a model in which CNK promotes Raf phosphorylation͞activation through membrane localization, oligomerization, or association with an activating kinase.scaffold ͉ vulva ͉ lin-45

A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila

“…CNK homologs have been identified in other metazoans and evidence gathered in mammalian cell lines supports their participation in the regulation of B-RAF and C-RAF (Lanigan et al 2003;Bumeister et al 2004;Ziogas et al 2005). A similar conclusion was also recently reached in C. elegans (Rocheleau et al 2005). In flies, CNK associates directly with the catalytic domain of RAF through a short amino acid sequence called the RAF-interacting motif (RIM) and modulates RAF activity according to the RTK signaling status (Douziech et al 2003;Laberge et al 2005).…”

The Caenorhabditis elegans ekl (Enhancer of ksr-1 Lethality) Genes Include Putative Components of a Germline Small RNA Pathway

“…Furthermore, when we tested our ekl RNAi clones against a panel of other Ras pathway mutants (materials and methods), including hypomorphic alleles of lin-45 raf and mek-2, we found that only 2 RNAi clones caused rod-like lethality in those backgrounds as would be expected if Ras signaling or the excretory duct fate were perturbed directly (data not shown); these corresponded to the known Ras pathway scaffold cnk-1 (Rocheleau et al 2005) and the GTPase rab-7 (L. Chotard and C. E. Rocheleau, unpublished results). The remaining 16 ekl RNAi clones showed rod-like lethality exclusively in the background of ksr-1 alleles, suggesting that these ekl genes influence a process to which ksr-1 mutants are particularly sensitive.…”

KSR and CNK: two scaffolds regulating RAS-mediated RAF activation

“…To verify its putative role in RAS signaling, a recent study used a reverse genetic approach and characterized cnk-1 deletion alleles that had been generated in an unbiased way (Rocheleau et al, 2005). Unlike in Drosophila, cnk-1 is not essential for viability and its elimination did not result in let-60-like phenotypes.…”