Signalling Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, U.K.
Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, U.K.
Biochem J. 2023 May 15;480(9):587-605. doi: 10.1042/BCJ20220598.
Innate or acquired resistance to small molecule BRAF or MEK1/2 inhibitors (BRAFi or MEKi) typically arises through mechanisms that sustain or reinstate ERK1/2 activation. This has led to the development of a range of ERK1/2 inhibitors (ERKi) that either inhibit kinase catalytic activity (catERKi) or additionally prevent the activating pT-E-pY dual phosphorylation of ERK1/2 by MEK1/2 (dual-mechanism or dmERKi). Here, we show that eight different ERKi (both catERKi or dmERKi) drive the turnover of ERK2, the most abundant ERK isoform, with little or no effect on ERK1. Thermal stability assays show that ERKi do not destabilise ERK2 (or ERK1) in vitro, suggesting that ERK2 turnover is a cellular consequence of ERKi binding. ERK2 turnover is not observed upon treatment with MEKi alone, suggesting it is ERKi binding to ERK2 that drives ERK2 turnover. However, MEKi pre-treatment, which blocks ERK2 pT-E-pY phosphorylation and dissociation from MEK1/2, prevents ERK2 turnover. ERKi treatment of cells drives the poly-ubiquitylation and proteasome-dependent turnover of ERK2 and pharmacological or genetic inhibition of Cullin-RING E3 ligases prevents this. Our results suggest that ERKi, including current clinical candidates, act as 'kinase degraders', driving the proteasome-dependent turnover of their major target, ERK2. This may be relevant to the suggestion of kinase-independent effects of ERK1/2 and the therapeutic use of ERKi.
对小分子 BRAF 或 MEK1/2 抑制剂(BRAFi 或 MEKi)的先天或获得性耐药通常通过维持或恢复 ERK1/2 激活的机制而产生。这导致了一系列 ERK1/2 抑制剂(ERKi)的开发,这些抑制剂要么抑制激酶催化活性(catERKi),要么另外阻止 MEK1/2 对 ERK1/2 的激活 pT-E-pY 双磷酸化(双机制或 dmERKi)。在这里,我们表明,八种不同的 ERKi(无论是 catERKi 还是 dmERKi)都会导致 ERK2(最丰富的 ERK 同工型)的周转,而对 ERK1 的影响很小或没有。热稳定性测定表明,ERKi 不会在体外使 ERK2(或 ERK1)不稳定,这表明 ERK2 的周转是 ERKi 结合的细胞后果。在用 MEKi 单独处理时不会观察到 ERK2 的周转,这表明是 ERKi 与 ERK2 的结合驱动了 ERK2 的周转。然而,MEKi 的预处理,它阻止了 ERK2 pT-E-pY 磷酸化和与 MEK1/2 的解离,阻止了 ERK2 的周转。ERK2 的聚泛素化和蛋白酶体依赖性周转是由 ERKi 处理细胞所驱动的,药理学或遗传抑制 Cullin-RING E3 连接酶可以防止这种情况发生。我们的结果表明,ERKi,包括当前的临床候选药物,作为“激酶降解剂”,驱动其主要靶点 ERK2 的蛋白酶体依赖性周转。这可能与 ERK1/2 的激酶非依赖性效应的建议以及 ERKi 的治疗用途有关。