Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA.
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
J Biol Chem. 2020 Jun 19;295(25):8494-8504. doi: 10.1074/jbc.RA119.012079. Epub 2020 May 5.
The selective pressure imposed by extrinsic death signals and stressors adds to the challenge of isolating and interpreting the roles of proteins in stress-activated signaling networks. By expressing a kinase with activating mutations and a caged lysine blocking the active site, we can rapidly switch on catalytic activity with light and monitor the ensuing dynamics. Applying this approach to MAP kinase 6 (MKK6), which activates the p38 subfamily of MAPKs, we found that decaging active MKK6 in fibroblasts is sufficient to trigger apoptosis in a p38-dependent manner. Both in fibroblasts and in a murine melanoma cell line expressing mutant B-Raf, MKK6 activation rapidly and potently inhibited the pro-proliferative extracellular signal-regulated kinase (ERK) pathway; to our surprise, this negative cross-regulation was equally robust when all p38 isoforms were inhibited. These results position MKK6 as a new pleiotropic signal transducer that promotes both pro-apoptotic and anti-proliferative signaling, and they highlight the utility of caged, light-activated kinases for dissecting stress-activated signaling networks.
外在的死亡信号和应激源所施加的选择压力增加了隔离和解释应激激活信号网络中蛋白质作用的难度。通过表达具有激活突变的激酶和一个封闭活性位点的笼状赖氨酸，我们可以用光快速开启催化活性，并监测随后的动力学。将这种方法应用于 MAP 激酶 6（MKK6），它激活 MAPK 的 p38 亚家族，我们发现，在成纤维细胞中去除活性 MKK6 足以 p38 依赖性方式触发细胞凋亡。在成纤维细胞和表达突变 B-Raf 的鼠黑素瘤细胞系中，MKK6 的激活迅速而有效地抑制了促有丝分裂的细胞外信号调节激酶（ERK）途径；令我们惊讶的是，当所有 p38 同工型都被抑制时，这种负交叉调节同样强大。这些结果将 MKK6 定位为一种新的多效信号转导器，它促进促凋亡和抗增殖信号，并且它们突出了笼状、光激活激酶在剖析应激激活信号网络中的效用。
