1. Centre for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, Ontario M5G 1X5, Canada
2. Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
3. Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling,
4. Département de pathologie et de biologie cellulaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada
5. These authors contributed equally to this work.

Correspondence to: Frank Sicheri^1,2Marc Therrien^3,4 Correspondence and requests for materials should be addressed to F.S. (Email: sicheri@lunenfeld.ca) or M.T. (Email: marc.therrien@umontreal.ca).

Abstract

The ERK (extracellular signal-regulated kinase) pathway is an evolutionarily conserved signal transduction module that controls cellular growth, differentiation and survival¹. Activation of receptor tyrosine kinases (RTKs) by the binding of growth factors initiates GTP loading of RAS, which triggers the initial steps in the activation of the ERK pathway by modulating RAF family kinase function. Once activated, RAF participates in a sequential cascade of phosphorylation events that activate MEK, and in turn ERK. Unbridled signalling through the ERK pathway caused by activating mutations in RTKs, RAS or RAF has been linked to several human cancers². Of note, one member of the RAF family, BRAF, is the most frequently mutated oncogene in the kinase superfamily³. Not surprisingly, there has been a colossal effort to understand the underlying regulation of this family of kinases. In particular, the process by which the RAF kinase domain becomes activated towards its substrate MEK remains of topical interest. Here, using Drosophila Schneider S2 cells, we demonstrate that RAF catalytic function is regulated in response to a specific mode of dimerization of its kinase domain, which we term the side-to-side dimer. Moreover, we find that the RAF-related pseudo-kinase KSR (kinase suppressor of Ras) also participates in forming side-to-side heterodimers with RAF and can thereby trigger RAF activation. This mechanism provides an elegant explanation for the longstanding conundrum about RAF catalytic activation, and also provides an explanation for the capacity of KSR, despite lacking catalytic function, to directly mediate RAF activation. We also show that RAF side-to-side dimer formation is essential for aberrant signalling by oncogenic BRAF mutants, and identify an oncogenic mutation that acts specifically by promoting side-to-side dimerization. Together, our data identify the side-to-side dimer interface of RAF as a potential therapeutic target for intervention in BRAF-dependent tumorigenesis.

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