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Abstract
Although extensively studied for three decades, the molecular mechanisms that regulate the RAF/MEK/ERK kinase cascade remain ambiguous. Recent studies identified the dimerization of RAF as a key event in the activation of this cascade. Here, we show that in-frame deletions in the β3-αC loop activate ARAF as well as BRAF and other oncogenic kinases by enforcing homodimerization. By characterizing these RAF mutants, we find that ARAF has less allosteric and catalytic activity than the other two RAF isoforms, which arises from its non-canonical APE motif. Further, these RAF mutants exhibit a strong oncogenic potential, and a differential inhibitor resistance that correlates with their dimer affinity. Using these unique mutants, we demonstrate that active RAFs, including the BRAF(V600E) mutant, phosphorylate MEK in a dimer-dependent manner. This study characterizes a special category of oncogenic kinase mutations, and elucidates the molecular basis that underlies the differential ability of RAF isoforms to stimulate MEK-ERK pathway. Further, this study reveals a unique catalytic feature of RAF family kinases that can be exploited to control their activities for cancer therapies.
Highlights
The Ras/rapidly-accelerated fibrosarcoma (RAF)/mitogenactivated protein kinase (MEK)/Extracellular signalregulated kinase (ERK) signaling plays a crucial role in Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.The RAF kinases CRAF, BRAF, and ARAF are a core component of the RAF/MEK/ERK kinase cascade
Similar to other constitutively-active mutants of RAF kinases, a co-expression of a dominant-negative RAS mutant (N17RAS) with ARAF(ΔQA) did not affect its activity in 293T cells (Fig. 1b), indicating that ARAF (ΔQA) is a constitutively-active mutant and does not require upstream stimuli to trigger its catalytic activity. This notion was further validated by the finding that a stable expression of ARAF(ΔQA) in wild-type, BRAF−/−, and CRAF−/− fibroblasts activated the MEK-ERK pathway and transformed cells independent of endogenous RAF molecules
The shRNAmediated knockdown of either CRAF in BRAF−/− fibroblasts or BRAF in CRAF−/− fibroblasts had no effect on the ability of ARAF(ΔQA) to stimulate downstream signaling (Figure S1B)
Summary
Dimerization among RAF isoforms is a key event in triggering the RAF/MEK/ERK kinase cascade [9,10,11,12,13,14,15,16,17,18], which turns on the kinase activity of RAF and facilitates the activation of MEK by RAF [19]. Mechanistic studies have shown that the two protomers play distinct roles in RAF dimers: one functions as an allosteric activator to facilitate the assembly of an active conformation in the other, which acts as a receiver to catalyze the phosphorylation of substrates [20]. Distinct molecular traits between BRAF and CRAF result in their differential ability to turn on the RAF/ MEK/ERK kinase cascade by dimerization-driven transactivation [21]. Whether ARAF can be activated by dimerization and its role in this process are unclear
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