The E3/E4 ubiquitin ligase UFD-2 suppresses normal and oncogenic signaling mediated by a Raf ortholog in Caenorhabditis elegans

Abstract

Signaling by the kinase cascade composed of Raf, MEK, and ERK is critical for animal development and is often inappropriately activated in human malignancies. We sought to identify factors that control signaling mediated by the Caenorhabditis elegans Raf ortholog LIN-45. A genetic screen showed that the degradation of LIN-45 required the E3/E4 ubiquitin ligase UFD-2. Both UFD-2 and its partner, the ATP-dependent segregase CDC-48, were required for the developmental regulation of LIN-45 protein abundance. We showed that UFD-2 acted in the same pathway as the E3 ubiquitin ligase SCF SEL-10 to decrease LIN-45 abundance in cells in which Raf-MEK-ERK signaling was most highly active. UFD-2 also reduced the protein abundance of activated LIN-45 carrying a mutation equivalent to the cancer-associated BRAF(V600E) variant. Our structure-function studies showed that the disruption of LIN-45 domains that mediate protein-protein interactions, including the conserved cysteine-rich domain and 14-3-3 binding motifs, were required for UFD-2–independent degradation of LIN-45. We propose a model in which UFD-2 and CDC-48 act downstream of SCF SEL-10 to remove LIN-45 from its protein interaction partners and facilitate proteasomal targeting and degradation. These findings imply that UFD-2 and CDC-48 may be important for Raf degradation during normal and oncogenic Ras and MAPK signaling in mammalian cells.

The Raf/LIN-45 C-terminal distal tail segment negatively regulates signaling inCaenorhabditis elegans

“…To test whether the LIN-45(S312A, L763stop) mutant can support signaling without wild-type lin-45(+), we introduced yfp-lin-45(S312A, L763stop) into the null mutant lin-45(dx19). When homozygous, this mutant is Vul, but can be completely rescued by yfp-lin-45(+) transgenes expressed in the VPCs (TOWNLEY et al 2023). In lin-45(dx19) mutants, we observed a highly penetrant Muv phenotype in animals expressing yfp-lin-45(S312A, L763stop) (Fig.…”

“…Residues Q95 and R118 of the LIN-45 RBD correspond to Q66 and R89 of RAF1, sites that are required for interaction with Ras-GTP and plasma membrane recruitment (BLOCK et al 1996;HSU et al 2002;HARDING et al 2003). We previously showed that transgenes expressing YFP-LIN-45(Q95A, R118A) fail to rescue the lin-45 null mutant, consistent with a loss of function (TOWNLEY et al 2023). To test whether a highly active LIN-45(S312A) form requires Ras binding, we generated the transgene yfp-lin-45(Q95A, R118A, S312A, Y810A).…”

“…To facilitate screening for negative regulators of C. elegans LIN-45, we produced strains carrying single-copy transgenes that express YFP-tagged LIN-45(S312A), a mutation that prevents phosphorylation at the residue equivalent to RAF1 S259. All transgenes made in C. elegans were constructed using a lin-31 promoter that drives expression in the VPCs during the L2 and L3 larval stages (MYERS AND GREENWALD 2005;TOWNLEY et al 2023). Two independent strains carrying wild-type yfplin-45(+) had normal development, with no adults displaying the Muv phenotype (TOWNLEY et al 2023) (Fig.…”

“…All transgenes made in C. elegans were constructed using a lin-31 promoter that drives expression in the VPCs during the L2 and L3 larval stages (MYERS AND GREENWALD 2005;TOWNLEY et al 2023). Two independent strains carrying wild-type yfplin-45(+) had normal development, with no adults displaying the Muv phenotype (TOWNLEY et al 2023) (Fig. 2a,b).…”

“…Mutations at two sites within the RAF1 CRD, R143E and T145E, disrupt its interaction with 14- 3-3 (CLARK et al 1997). We previously showed that mutations in the T177E), cause weak gain-of-function activity in the lin-45(dx19) null mutant background (TOWNLEY et al 2023). In BRAF, a single missense change at the same site, BRAF(T241P), is associated with CFC (SCHULZ et al 2008).…”