Functional analysis of CNK in RAS signaling.

Connector enhancer of KSR (CNK) is a multidomain protein required for RAS signaling. Its C-terminal portion (CNK(C-term)) directly binds to RAF. Herein, we show that the N-terminal portion of CNK (CNK(N-term)) strongly cooperates with RAS, whereas CNK(C-term) efficiently blocks RAS- and RAF-dependent signaling when overexpressed in the Drosophila eye. Two effector loop mutants of RAS(V12), S35 and C40, which selectively activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase pathways, respectively, do not cooperate with CNK. However, a strong cooperation is observed between CNK and RAS(V12G37), an effector loop mutant known in mammals to activate specifically the RAL pathway. We have identified two domains in CNK(N-term) that are critical for cooperation with RAS. Our results suggest that CNK functions in more than one pathway downstream of RAS. CNK(c-term) seems to regulate RAF, a component of the MAPK pathway, whereas CNK(N-term) seems to be involved in a MAPK-independent pathway.