Mutation that blocks ATP binding creates a pseudokinase stabilizing the scaffolding function of kinase suppressor of Ras, CRAF and BRAF

Abstract

Because mutations in RAS and BRAF represent the most common mutations found in human tumors, identification of inhibitors has been a major goal. Surprisingly, new oncogenic BRAF specific inhibitors inhibit cells transformed with mutated BRAF but paradoxically stimulate the growth of cells transformed with RAS. Here, we show that the mechanism for activation is via drug-induced dimer formation between CRAF and kinase suppressor of Ras (KSR)1. To understand the function of KSR1, we generated a KSR1 mutant that cannot bind ATP but stabilizes the closed, active conformation of KSR1. Molecular modeling suggested that the mutant stabilizes the two hydrophobic spines critical for the closed active conformation. We, therefore, could use the mutant to discriminate between the scaffold versus kinase functions of KSR1. The KSR1 mutant bound constitutively to RAF and mitogen-activated protein kinase kinase (MEK) but could not reconstitute activity suggesting that the catalytic activity of KSR1 is required for its function. Analogous mutations in BRAF and CRAF allowed us to test the generality of the model. The mutation induced changes consistent with the active, closed conformation of both kinases and confirmed that BRAF functions distinctly from CRAF in the MAP kinase pathway. Not only does this work suggest that KSR1 may function as a kinase, we anticipate that the mutation that we generated may be broadly applicable to stabilize the closed conformation of other kinases many of which may also form dimers.cancer | protein kinase | signal transduction

Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions characterizing pediatric astrocytomas

“…Recently, a more prominent role for kinase suppressor of RAS (KSR) in regulating RAF phosphorylation of MEK that also implicates KSR-BRAF heterodimerization has been suggested (14). Furthermore, in a separate study, KSR1 was proposed to compete with CRAF for inhibitor-induced binding to BRAF and alter paradoxical activation induced by BRAF inhibition (15,16). Although CRAF does not appear to play a role in the drug resistance or paradoxical activation associated with KIAA1549-BRAF signaling, we tested whether the fusion kinase displays altered or differential interactions with KSR1.…”

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

“…5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 11 The role of KSR1 as a RAF dimerization competitor has been proposed in several studies before. In one study it has been shown that KSR1 competes with C-RAF for inhibitorbound B-RAF leading to attenuation of ERK activation [18], whereas in another study KSR1 was found to be required for the inhibitor-induced hyperactivation through promoting KSR1:C-RAF dimerization [17]. More recently, it has been shown that the affinity of the oncogenic KIAA1549-BRAF fusion protein found in astrocytomas to KSR1 appeared to be much higher than that of B-RAF wild type or B-RAF-V600E [23].…”

“…At that time, when the KSR1 function was considered only in scaffolding of MAPK components, the loss of signalling at supra-physiologic KSR1 expression levels was proposed to be due to the titration of pathway members away from each other, a phenomenon termed as "combinatorial inhibition" [54]. However, emerging evidence suggests that KSR1 possesses dual activity as a scaffold protein and maybe also as active kinase phosphorylating C-RAF and MEK [17,[57][58][59]. Moreover, KSR1 can also form dimers with RAF and modulate its activity adding complexity to the simple view of KSR1 scaffolding function [17][18].…”

“…However, emerging evidence suggests that KSR1 possesses dual activity as a scaffold protein and maybe also as active kinase phosphorylating C-RAF and MEK [17,[57][58][59]. Moreover, KSR1 can also form dimers with RAF and modulate its activity adding complexity to the simple view of KSR1 scaffolding function [17][18]. In light of these findings, our data suggest that the peculiar concentration-dependent effect of DiRas3 on Ras12V-iduced transformation may be due to involvement of KSR1, the protein whose function and regulation is barely understood.…”

“…Interestingly, some of the RAF inhibitors do not efficiently induce B-RAF:C-RAF dimers but can still activate MEK and ERK in Ras transformed cells [14,19,25]. 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].…”