Functional analysis of CNK in RAS signaling

Connector enhancer of KSR (CNK) is a multidomain protein required for RAS s ignaling. Its C-terminal portion (CNKC-term) directly binds to RAF. Herein, we show that the N-terminal portion of CNK (CNKN-term) strongly cooperates with RAS, whereas CNKC-term efficiently blocks RAS- and RAF-dependent sign aling when overexpressed in the Drosophila eye. Two effector loop mutants o f RAS(V12), S35 and C40, which selectively activate the mitogen-activated p rotein kinase (MAPK) and phosphatidylinositol-3-kinase pathways, respective ly, do not cooperate with CNK. However, a strong cooperation is observed be tween CNK and RAS(V12G37), an effector loop mutant known in mammals to acti vate specifically the RAL pathway. We have identified two domains in CNKN-t erm that are critical for cooperation with RAS. Our results suggest that CN K functions in more than one pathway downstream of RAS. CNKC-term seems to regulate RAF, a component of the MAPK pathway, whereas CNKN-term Seems to b e involved in a MAPK-independent pathway.