Sci. Signal., 26 February 2013
Protein Interactions Targeting Dimerization
Nancy R. Gough
Science Signaling, AAAS, Washington, DC 20005, USA
Mutations in proteins of and aberrant signaling through the Ras-Raf–mitogen-activated protein kinase (MAPK) cascade are responsible both for genetic diseases and for many cancers. There are three members of the Raf kinase family, A-Raf, B-Raf, and C-Raf. A-Raf is the least active of these, and mutants of B-Raf and C-Raf have been implicated in several pathological conditions. These kinases are activated by interaction with Ras, which promotes their dimerization. Freeman et al. performed coimmunoprecipitation assays with endogenous and tagged forms of the Raf isoforms and determined that, in response to growth factor signaling, B-Raf and C-Raf interacted with one another, and B-Raf and A-Raf interacted with one another, but A-Raf and C-Raf did not. Selective depletion of specific isoforms showed that decreasing A-Raf did not alter the activity of growth factor–stimulated B- or C-Raf but that B-Raf in the absence of C-Raf or C-Raf in the absence of B-Raf resulted in compromised activation. Analysis of R>H mutants (with R to H mutations at the dimerization interface of both C-Raf and B-Raf) or E>K mutants (with E to K mutations at the dimerization interface of both C-Raf and B-Raf) showed that the R>H mutations exhibited compromised heterodimerization, whereas the E>K set showed increased heterodimerization and increased basal activity. The E>K mutant of C-Raf showed a substantial increase in growth factor–induced homodimerization and activity, whereas there was little or no change in activity and homodimerization of the E>K B-Raf mutant. These mutations were also introduced into disease-associated mutant forms of Raf with high, moderate, or low activity, and in all cases the R>H mutation disrupted B- and C-Raf heterodimerization and the E>K mutation enhanced heterodimerization. In a focus formation assay, the R>H mutation reduced the number of foci for cells expressing all but the B-Raf mutants with high catalytic activity, and the E>K mutation increased foci formation. Targeting this dimerization interface with a peptide blocked growth factor–stimulated B- and C-Raf heterodimerization and downstream signaling of the wild-type proteins and all tested disease-causing mutants except the high-activity B-Raf mutants. Functionally, the peptide effectively reduced proliferation and viability of multiple non–small cell lung carcinoma cell lines—which have either low activity B-Raf mutations or activated Ras—but was not effective in A375 melanoma cells that have a high-activity B-Raf mutation. Thus, the dimerization interface may be an effective therapeutic target for diseases associated with increased Ras-Raf signaling, except in the cases with high-activity B-Raf mutations.
A. K. Freeman, D. A. Ritt, D. K. Morrison, Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling. Mol. Cell 49, 751–758 (2013). [PubMed]
Citation: N. R. Gough, Targeting Dimerization. Sci. Signal. 6, ec51 (2013).
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