Fatemeh Cheraghi
University of Wisconsin-Milwaukee
Milwaukee, WI
Using Caenorhabditis elegans to test the impact of 14-3-3 on Raf protein levels and localization
Fatemeh Cheraghi
Noonan Syndrome (NS) is a developmental disorder associated with increased Ras signaling. Gain-of-function mutations in RAF1, a member of Raf family kinases, are found in ~5% of NS patients. Raf kinases contain an N-terminal regulatory domain responsible for Ras binding and kinase autoinhibition. A second region, termed CR2, is a phosphorylated motif that interacts with 14-3-3 proteins. A C-terminal kinase domain is responsible for catalytic activity. The CR2 motif and a second 14-3-3 site, located near the C-terminus, promotes 14-3-3 binding and kinase autoinhibition. In NS patients, many RAF1 mutations are clustered at or around the CR2 14-3-3 binding site. Although many NS mutations result in increased kinase activity, others do not. We used Caenorhabditis elegans to assess the impact on Raf/LIN-45 activity, protein abundance, and localization when either of the conserved 14-3-3-binding sites is mutated. We used CRISPR/Cas9 techniques to generate alleles that disrupt the CR2 and the C-terminal 14-3-3 binding sites. Mutation of the CR2 site did not cause obvious phenotypes. In contrast, mutation of the C-terminal site resulted in a vulvaless phenotype characteristic of Raf/LIN-45 loss of function. In a double mutant lacking both CR2 and C-terminal sites, vulvaless phenotype was partially suppressed. These results are consistent with a model where the C-terminal site contributes to Raf activation, and the CR2 site contributes to Raf inhibition. In future studies, we will test how Raf/LIN-45 levels and localization are affected by 14-3-3 binding. The results will contribute to understanding of Raf regulation by 14-3-3 proteins.
SACNAS National Diversity in STEM Conference, Phoenix, AZ, October 30-November 2, 2024
