Drosophila Raf's N Terminus Contains a Novel Conserved Region and Can Contribute to Torso RTK Signaling

Drosophila Raf (DRaf) contains an extended N terminus, in addition to three conserved regions (CR1-CR3); however, the function(s) of this N-terminal segment remains elusive. In this article, a novel region within Draf's N terminus that is conserved in BRaf proteins of vertebrates was identified and termed conserved region N-terminal (CRN). We show that the N-terminal segment can play a positive role(s) in the Torso receptor tyrosine kinase pathway in vivo, and its contribution to signaling appears to be dependent on the activity of Torso receptor, suggesting this N-terminal segment can function in signal transmission. Circular dichroism analysis indicates that DRaf's N terminus (amino acids 1-117) including CRN (amino acids 19-77) is folded in vitro and has a high content of helical secondary structure as predicted by proteomics tools. In yeast two-hybrid assays, stronger interactions between DRaf's Ras binding domain (RBD) and the small GTPase Ras1, as well as Rap1, were observed when CRN and RBD sequences were linked. Together, our studies suggest that DRaf's extended N terminus may assist in its association with the upstream activators (Ras1 and Rap1) through a CRN-mediated mechanism(s) in vivo.