Abstract
Raf protein kinases act as Ras-GTP sensing components of the ERK signal transduction pathway in animal cells, influencing cell proliferation, differentiation, and survival. In humans, somatic and germline mutations in the genes BRAF and RAF1 are associated with malignancies and developmental disorders. Recent studies shed light on the structure of activated Raf, a heterotetramer consisting of Raf and 14-3-3 dimers, and raised the possibility that a Raf C-terminal distal tail segment (DTS) regulates activation. We investigated the role of the DTS using the Caenorhabditis elegans Raf ortholog lin-45. Truncations removing the DTS strongly enhanced lin-45(S312A), a weak gain-of-function allele equivalent to RAF1 mutations found in patients with Noonan Syndrome. We genetically defined three elements of the LIN-45 DTS, which we termed the active site binding sequence (ASBS), the KTP motif, and the aromatic cluster. In the context of lin-45(S312A), the mutation of each of these elements enhanced activity. We used AlphaFold to predict DTS protein interactions for LIN-45, fly Raf, and human BRAF within the activated heterotetramer complex. We propose the following distinct functions for the LIN-45 DTS elements: (1) the ASBS binds the kinase active site as an inhibitor; (2) phosphorylation of the KTP motif modulates the DTS-kinase domain interaction; and (3) the aromatic cluster anchors the DTS in an inhibitory conformation. Human RASopathy-associated variants in BRAF affect residues of the DTS, consistent with these predictions. This work establishes that the Raf/LIN-45 DTS negatively regulates signaling in C. elegans and provides a model for its function in other Raf proteins.