Bimodal regulation of RAF by CNK in Drosophila.

Connector enhancer of KSR (CNK) is a multidomain-containing protein previously identified as a positive regulator of the RAS/MAPK pathway in Drosophila. Using transfection experiments and an RNAi-based rescue assay in Drosophila S2 cells, we demonstrate that CNK has antagonistic properties with respect to RAF activity. We show that CNK's N-terminal region contains two domains (SAM and CRIC) that are essential for RAF function. Unexpectedly, we also report that the C-terminal region of CNK contains a short bipartite element that strongly inhibits RAF catalytic function. Interestingly, CNK's opposite properties appear to prevent signaling leakage from RAF to MEK in the absence of upstream signals, but then transforms into a potent RAF activator upon signal activation. Together, these findings suggest that CNK not only participates in the elusive RAF activation process, but might also contribute to the switch-like behavior of the MAPK module.