A tail of three regulatory elements: insights into the structure of C. elegans Raf/LIN-25

Mutations that affect Raf protein function cause human diseases, including cancer. Since the Raf protein pathway is conserved in the Caenorhabditis elegans nematode, genetic tools in the extensively studied worm allow us a window into how things work. It’s axiomatic that structure dictates function, and thus altering a protein’s structure—including its binding domains—changes how it acts. 

New insights into the structure and function of C. elegans Raf were published in the November 2024 issue of GENETICS. Robert A. Townley and colleagues from the de la Cova lab at the University of Wisconsin–Milwaukee investigated the role of the distal tail segment (DTS) in the activation of LIN-45, the worm Raf ortholog.

Raf protein kinases serve as Ras-GTP sensors in the MAPK/ERK signal transduction pathway. When cells receive growth signals, Raf is activated by Ras-GTP formation, dimerizing and then forming a heterotetramer with 14-3-3. This drives the MAPK/ERK cascade, leading to the expression of genes that promote cell growth, differentiation, and survival—and also results in a negative feedback loop that turns Raf back off again. 

Humans have three genes encoding Raf, and mutations in two of those genes–BRAF and RAF1–are associated with tumor development and RASopathy disorders like Noonan Syndrome. Although individual RASopathies are rare, they collectively cause most genetics-related learning and development problems.

Previous studies clarified the structure of activated Raf and suggested that a Raf C-terminal DTS negatively regulates activation. To further the understanding of Raf structure and function, Townley et al. did a mutational deep dive into the DTS, assessing how specific mutations at particular residues affected vulval development as a readout of Raf activity. They used AlphaFold to predict the structure of LIN-45 bound to PAR-5 (the worm 14-3-3 ortholog) and to predict what effects various residue changes would have. Again using AlphaFold, the researchers compared two isoforms of human BRAF, Drosophila Raf, and LIN-45. They identify roles for distinct elements within the DTS, which overall act to negatively regulate Raf activation.

The work by Townley et al. establishes the DTS’s function in activation by determining that its removal greatly increases the activity of lin-45(S312A), a weak gain-of-function allele equivalent to RAF1 mutations in Noonan Syndrome patients. 

The authors were able to genetically define three regions of the LIN-45 DTS: the active site binding sequence (ASBS), the KTP motif, and the aromatic cluster. They used AlphaFold to predict the structure of heterotetramers containing LIN-45 and the 14-3-3 worm ortholog PAR-5; this modeling allowed them to interrogate the effects of changing a number of individual residues in the domains of interest. They also used AlphaFold to compare predicted structures for LIN-45, Drosophila Raf, and human BRAF. The authors propose that the ASBS inhibits kinase action, the phosphorylated KTP motif modulates DNS-kinase interaction, and the aromatic cluster secures the DTS’s inhibitory conformation. 

In line with the AlphaFold predictions, the researchers found that human RASopathy-associated variants in BRAF affect DTS residues. Along with establishing that the Raf/LIN-45 DTS negatively regulates signaling in C. elegans, this work offers a model for the DTS function in other Raf proteins, including those involved in cancer formation.