A dimerization-dependent mechanism drives RAF catalytic activation

Abstract

The ERK (extracellular signal-regulated kinase) pathway is an evolutionarily conserved signal transduction module that controls cellular growth, differentiation and survival. 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 cancers. Of note, one member of the RAF family, BRAF, is the most frequently mutated oncogene in the kinase superfamily. 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.

Phosphorylation of RAF Kinase Dimers Drives Conformational Changes that Facilitate Transactivation

“…Remarkably, the C‐terminal end of the αC‐helix is neutral in most kinases, whereas in RAFs it carries a +3 charge. Moreover, mutations of these charged residues impair kinase activity14 and affect RAF homo‐ and heterodimerization 3b, 14, 15. Since RAF kinases dimerize via the C‐terminal end of the αC‐helix, the accumulation of six positive charges at the dimerization interface enables interaction with the highly negatively charged NtA motif (specific to RAF), thereby promoting the dimerization.…”

Stabilization of C-RAF:KSR1 complex by DiRas3 reduces availability of C-RAF for dimerization with B-RAF

“…5D), which efficiently hinders the A-RAF:B-RAF heterodimerization [46]. Furthermore, although the side-to-side dimerization mode also applies to the heterodimerization between RAF kinases and the structurally related KSR proteins [20], we observed, to our surprise, that DiRas3 did not inhibit the RAF:KSR1 dimerization but rather enhanced it. These results disprove the hypothesis that DiRas3 hinders RAF heterodimerization by direct binding to the dimerization interface.…”

“…Previous studies have shown that RAF kinases dimerize also with the structurally related KSR proteins (KSR1 and KSR2) to form side-to-side RAF:KSR heterodimers [20]. Therefore, we assumed that DiRas3 may also inhibit the dimerization of RAF proteins with KSR.…”

“…These drugs induce dimerization between C-RAF and the structurally related KSR1 (kinase suppressor of Ras 1) [17]. Indeed, based on the symmetric packing of RAF molecules in the crystal structures, it has been suggested that the side-to-side dimer interface, conserved in KSR and in all RAF isoforms, mediates the ability of RAF to bind to itself or to KSR [20].…”

Régulation de la survie cellulaire par les kinases de la famille RAF

“…De plus, certaines phosphorylations régulent indirectement l'activité kinase en changeant l'interaction des RAF avec d'autres protéines : en particulier, les protéines de la famille 14-3-3 qui reconnaissent des ligands phosphorylés et interagissent avec les kinases RAF. Elles permettent la formation de dimères entre les différentes kinases RAF, ce qui constitue une modalité importante de leur régulation [13]. À ce jour, les kinases MEK1/2, qui font partie du complexe d'activation de RAF, constituent l'unique substrat direct connu de ces kinases.…”