RAF Isoforms Have Different Roles in Kinase Transactivation

RAF kinases are a critical component of the RAS signaling cascade and stimulate downstream activation of MEK/ERK signaling. However, although RAF activation has been suggested to involve recruitment to the plasma membrane, dimerization of RAF monomers, and RAF phosphorylation, the mechanism of RAF activation is not fully understood. In addition, it remains unclear why kinase-inactive variants of BRAF but not CRAF are oncogenic and can activate MEK/ERK signaling. To better understand the mechanisms that control RAF activation, Hu and colleagues generated dimers consisting of an active RAF kinase, referred to as the receiver, and a kinase-dead RAF variant, referred to as the activator. RAF transactivation was dependent on dimerization, which was mediated in part by a conserved tryptophan residue at the dimer interface, and on phosphorylation of the activator monomer within an N-terminal acidic (NtA) motif; this modification occurs constitutively in BRAF but not CRAF, explaining the distinct ability of kinase-dead BRAF to transactivate CRAF. In contrast, phosphorylation of the CRAF NtA motif was stimulated by MEK, supporting the presence of a positive feedback loop that converts CRAF into an activator kinase following its transactivation by BRAF. Dimerization and phosphorylation of the activator NtA motif allosterically induced cis-autophosphorylation of the receiver kinase within its activation loop, suggesting that these events facilitate assembly and stabilization of the active kinase conformation and prime CRAF for autophosphorylation. Consistent with this idea, mutation of receiver monomers within the hydrophobic regulatory spine, which is critical for active kinase assembly, bypassed the requirement for dimerization and phosphorylation and resulted in constitutive RAF kinase activity. Together, these results show that RAF dimers are functionally asymmetric and provide insight into the complex mechanisms underlying RAF transactivation.

Hu J, Stites EC, Yu H, Germino EA, Meharena HS, Stork PJ, et al. Allosteric activation of functionally asymmetric RAF kinase dimers. Cell 2013;154:1036–46.