Membrane localization of Raf assists engagement of downstream effecters

We have previously described a small molecule-directed protein dimerization strategy, using coumermycin to juxtapose Raf fusion proteins containing th e coumermycin-binding domain GyrB. Oligomerization of cytoplasmically local ized Raf-GyrB fusion proteins leads to an increase in the kinase activity o f both Raf and its substrate Mek. Surprisingly, more distal targets, such a s Erk1 and Erk2, are not activated using this approach. Here we report that coumermycin-induced oligomerization of a membrane-localized Raf-GyrB fusio n protein potently activated Erk1 and Erk2, up-regulated Fos protein levels , and induced expression of many immediate-early response genes. Thus, both membrane localization and oligomerization of Raf-GyrB are required to targ et Raf signals to downstream effecters. The ability to activate the entire Raf signal transduction cascade conditionally, using coumermycin-induced ol igomerization, should prove useful for dissecting Raf-mediated effects on g ene expression and cellular differentiation.