Department of Biological Sciences, The University of Toledo, Toledo, OH 43606, USA.
Department of Biological Sciences, The University of Toledo, Toledo, OH 43606, USA.
Cell Signal. 2017 Nov;39:66-73. doi: 10.1016/j.cellsig.2017.07.021. Epub 2017 Jul 28.
Mixed Lineage Kinase 3 (MLK3), a member of the MLK subfamily of protein kinases, is a mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) that activates MAPK signalling pathways and regulates cellular responses such as proliferation, invasion and apoptosis. MLK4β, another member of the MLK subfamily, is less extensively studied, and the regulation of MLK4β by stress stimuli is not known. In this study, the regulation of MLK4β and MLK3 by osmotic stress, thermostress and heat shock protein 90 (Hsp90) inhibition was investigated in ovarian cancer cells. MLK3 and MLK4β protein levels declined under conditions of prolonged osmotic stress, heat stress or exposure to the Hsp90 inhibitor geldanamycin (GA); and MLK3 protein declined faster than MLK4β. Similar to MLK3, the reduction in MLK4β protein in cells exposed to heat or osmotic stresses occurred via a mechanism that involves the E3 ligase, carboxy-terminus of Hsc70-interacting protein (CHIP). Both heat shock protein 70 (Hsp70) and CHIP overexpression led to polyubiquitination and a decrease in endogenous MLK4β protein, and MLK4β was ubiquitinated by CHIP in vitro. In untreated cells and cells exposed to osmotic and heat stresses for short time periods, small interfering RNA (siRNA) knockdown of MLK4β elevated the levels of activated MLK3, c-Jun N-terminal kinase (JNK) and p38 MAPKs. Furthermore, MLK3 binds to MLK4β, and this association is regulated by osmotic stress. These results suggest that in the early response to stressful stimuli, MLK4β-MLK3 binding is important for regulating MLK3 activity and MAPK signalling, and after prolonged periods of stress exposure, MLK4β and MLK3 proteins decline via CHIP-dependent degradation. These findings provide insight into how heat and osmotic stresses regulate MLK4β and MLK3, and reveal an important function for MLK4β in modulating MLK3 activity in stress responses.
混合谱系激酶 3（MLK3）是丝裂原活化蛋白激酶（MAPK）激酶激酶（MAP3K）家族的一个成员，它激活 MAPK 信号通路并调节细胞反应，如增殖、侵袭和凋亡。MLK4β是 MLK 亚家族的另一个成员，其研究较少，应激刺激对 MLK4β 的调节尚不清楚。在这项研究中，研究了渗透压应激、热应激和热休克蛋白 90（Hsp90）抑制对卵巢癌细胞中 MLK4β 和 MLK3 的调节。在渗透压应激、热应激或暴露于 Hsp90 抑制剂格尔德霉素（GA）的情况下，MLK3 和 MLK4β 的蛋白水平下降；并且 MLK3 蛋白下降速度快于 MLK4β。与 MLK3 相似，暴露于热或渗透压应激的细胞中 MLK4β 蛋白的减少是通过 E3 连接酶羧基末端的 Hsc70 相互作用蛋白（CHIP）的机制发生的。热休克蛋白 70（Hsp70）和 CHIP 的过表达导致内源性 MLK4β 蛋白的多泛素化和减少，并且 MLK4β 在体外被 CHIP 泛素化。在未处理的细胞和暴露于渗透压和热应激的细胞中，短时间内，MLK4β 的小干扰 RNA（siRNA）敲低可提高激活的 MLK3、c-Jun N-末端激酶（JNK）和 p38 MAPKs 的水平。此外，MLK3 与 MLK4β 结合，这种结合受渗透压应激调节。这些结果表明，在应激刺激的早期反应中，MLK4β-MLK3 结合对于调节 MLK3 活性和 MAPK 信号转导很重要，并且在长时间暴露于应激后，MLK4β 和 MLK3 蛋白通过 CHIP 依赖性降解而下降。这些发现提供了关于热和渗透压应激如何调节 MLK4β 和 MLK3 的见解，并揭示了 MLK4β 在应激反应中调节 MLK3 活性的重要功能。
