Oncogenic Raf signaling
In humans, a large proportion of melanomas harbor an oncogenic mutation, BRAF(V600E), which activates Raf signaling. While most melanomas develop from benign nevi (commonly known as moles), it is notable that moles with the BRAF(V600E) mutation rarely display increased Raf signaling. This suggests that they employ inhibitory mechanisms to prevent oncogenic Raf signaling.
Genetic screens in C. elegans
Our approach to investigate Raf signaling uses the roundworm Caenorhabditis elegans. The C. elegans genome encodes one Raf ortholog, termed LIN-45.
We performed genetic screens to identify cellular mechanisms that prevent signaling by a LIN-45 mutant modeled on BRAF(V600E), termed lin-45(V627E). Using a forward genetic screen, we discovered that the E3/E4 ubiquitin ligase UFD-2 promotes LIN-45 degradation, and UFD-2 loss greatly exacerbates phenotypes caused by lin-45(V627E) (Townley, et al. 2023). In a second approach, we used a targeted screen of conserved kinases, discovering that ERK, CDK2, and GSK3 promote LIN-45 degradation (de la Cova, et al. 2020).
Mutations in RASopathies
In humans, inherited and de novo germline mutations that alter Raf are associated with RASopathies. For example, 5% of Noonan syndrome patients carry mutations in the RAF1 gene, and 75% of patients diagnosed with Cardiofaciocutaneous syndrome carry mutations in BRAF. Although RAF1 and BRAF gene variants detected in RASopathy patients are often assumed to be significant, many are unique and their molecular consequences are unknown.
Disease variants in C. elegans
C. elegans are transparent, allowing us to visualize endogenous LIN-45 protein in living animals. In this context, we use CRISPR/Cas9 methods to generate mutations equivalent to those found in human RAF1 and BRAF genes. In C. elegans mutants, we assess the effect on LIN-45 protein abundance, localization, and activity.
Live visualization of ERK signaling
In humans and C. elegans, cell signaling by Raf relies on dynamic events, such as production of growth factors, binding to receptors, and activation of Raf, MEK, and ERK kinases. Individual cells can adopt distinct responses, dependent on the tissue type and receptors expressed.
To assess Raf-MEK-ERK signaling in living animals, we employ the GFP-tagged biosensor ERK-KTR, which responds to ERK activity through a change in its subcellular distribution (de la Cova et al. 2017).
Our recent work (Rodriguez Torres, et al. 2024) used the ERK-KTR to investigate Fibroblast growth factor (FGF) signaling in two cell types, muscle precursors and skin, and we analyzed the effects of mutations in the C. elegans FGFR.