RESUMEN
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), 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 distinct functions for the LIN-45 DTS elements: i) the ASBS binds the kinase active site as an inhibitor, ii) phosphorylation of the KTP motif modulates DTS-kinase domain interaction, and iii) 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.
RESUMEN
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, which has a single Raf ortholog termed lin-45 . We discovered that 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 generated mutations to test 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), mutation of either the ASBS, KTP motif, or aromatic cluster 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 distinct functions for the LIN-45 DTS elements: i) the ASBS binds the kinase active site as an inhibitor, ii) phosphorylation of the KTP motif modulates DTS-kinase domain interaction, and iii) the aromatic cluster anchors the DTS in an inhibitory conformation. 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.
RESUMEN
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 SCFSEL-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 SCFSEL-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.