Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling
17.04.2012
Röring M, Herr R, Fiala GJ, Heilmann K, Braun S, Eisenhardt AE, Halbach S, Capper D, von Deimling A, Schamel WW, Saunders DN, Brummer T.
EMBO J. 2012; 31(11):2629-47
The dimerisation of Raf kinases involves a central cluster within the kinase domain, the dimer interface (DIF). Yet, the importance of the DIF for the signalling potential of wild-type B-Raf (B-Raf wt) and its oncogenic counterparts remains unknown. Here, we show that the DIF plays a pivotal role for the activity of B-Raf wt and several of its gain-of-function (g-o-f) mutants. In contrast, the B-RafV600E, B-RafinsT and B-RafG469A oncoproteins are remarkably resistant to mutations in the DIF. However, compared with B-Rafwt, B-RafV600E displays extended protomer contacts, increased homodimerisation and incorporation into larger protein complexes. In contrast, B-Rafwt and Raf-1wt mediated signalling triggered by oncogenic Ras as well as the paradoxical activation of Raf-1 by kinase-inactivated B-Raf require an intact DIF. Surprisingly, the B-Raf DIF is not required for dimerisation between Raf-1 and B-Raf, which was inactivated by the D594A mutation, sorafenib or PLX4720. This suggests that paradoxical MEK/ERK activation represents a two-step mechanism consisting of dimerisation and DIF-dependent transactivation. Our data further implicate the Raf DIF as a potential target against Ras-driven Raf-mediated (paradoxical) ERK activation.
