It Takes Two to Make a RAF Signal Right

Advances in genomic technologies have allowed the credentialing of novel therapeutic targets defined on the basis of new knowledge about cancer genetics. Commensurate advances in molecular-resolution imaging may be needed to enable more precise therapies directed against these molecularly defined targets. Now, Nan et al. report on a new fluorescence-imaging and spacial analysis methodology used to directly visualize cell membrane–localized multimers of CRAF, a RAF-family signaling protein known to be involved in cancer progression. Activating oncogenic mutations in the RAS-RAF-MEK-ERK signaling pathway have been observed with high frequency across many cancers, and intensive research has been directed to develop pharmacological strategies to inhibit pathway components. However, as with most antineoplastic drug-discovery efforts, some potential therapeutic targets are currently considered “undruggable” (for example, RAS), and cancer cells adeptly acquire compensatory mutations to evade therapies directed at other targets (for example, BRAF). Technologies that enable enhanced single-molecule resolution of oncogenic signaling could be used to devise high-resolution functional screens for molecules that disrupt such process or to guide rational drug design strategies. With the new methodology—called photoactivated localization microscopy (PALM)—a protein of interest is attached to a fluorescence protein that is photoactivated so as to form an isolated image on a detector, allowing localization within 10 to 20 nm. The authors developed a new computational spacial analysis method to assess, for a given protein, the proportion of monomers and multimers that exist in a population, along with their subcellular locations. As proof of principle, the authors analyzed cells expressing an artificial dimer of the PAmCherry1 fluorescence protein and detected 74% of the protein in dimers, compared with only 6% of proteins detected as dimers in cells expressing PAmCherry1 monomers. Analysis of KRASWT cells transfected with a PAmCherry1-CRAF expression construct demonstrated that CRAF proteins existed predominantly as cytosolic monomers. However, coexpression of the constitutively active KRASG12D mutant isoform caused a 26-fold increase in the density of membrane-associated CRAF dimers and higher-order multimers. The authors further demonstrated that truncation of the auto-inhibitory N-terminal domain of CRAF proteins caused RAS-independent dimerization and phosphorylation of ERK, both of which were ablated by mutations in the CRAF dimerization interface. The C-terminal RAS CAAX motif has been demonstrated to govern clustering of membrane-associated RAS. Thus, CRAF-CAAX chimeric proteins displayed membrane-associated clustering similar to that of CRAF coexpressed with KRASG12D. In this case, mutation of the CRAF dimerization interface reduced ERK activation but did not alter the clustering of membrane-associated CRAF, suggesting a dominant role for the CAAX motif (and likely KRAS) in CRAF multimerization. Insights into the molecular mechanisms governing key signaling processes could enable the development of more precise therapies designed to counteract their pathophysiological dysregulation. For example, the work of Nan et al . suggests uses, in drug discovery, for assays that detect disruption of RAF dimerization as a functional readout of RAF inhibition or for the rational design of drugs that target the RAF dimerization interface. In general, the new work illustrates that advances in molecular-resolution imaging technologies may be needed to translate the genomic revolution into a drug-discovery one. X. Nan et al .,Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling. Proc. Natl. Acad. Sci. U.S.A. , published online 24 October 2013 (10.1073/pnas.1318188110). [[Abstract]][1] [1]: http://www.pnas.org/content/early/2013/10/23/1318188110.abstract