Caunt Christopher J, Armstrong Stephen P, Rivers Caroline A, Norman Michael R, McArdle Craig A
Laboratory for Integrated Neuroscience and Endocrinology, Department of Clinical Sciences at South Bristol, University of Bristol, Whitson Street, Bristol BS1 3NY, United Kingdom.
J Biol Chem. 2008 Sep 26;283(39):26612-23. doi: 10.1074/jbc.M801500200. Epub 2008 Jul 23.
Although many stimuli activate extracellular signal-regulated kinases 1 and 2 (ERK1/2), the kinetics and compartmentalization of ERK1/2 signals are stimulus-dependent and dictate physiological consequences. ERKs can be inactivated by dual specificity phosphatases (DUSPs), notably the MAPK phosphatases (MKPs) and atypical DUSPs, that can both dephosphorylate and scaffold ERK1/2. Using a cell imaging model (based on knockdown of endogenous ERKs and add-back of wild-type or mutated ERK2-GFP reporters), we explored possible effects of DUSPs on responses to transient or sustained ERK2 activators (epidermal growth factor and phorbol 12,13-dibutyrate, respectively). For both stimuli, a D319N mutation (which impairs DUSP binding) increased ERK2 activity and reduced nuclear accumulation. These stimuli also increased mRNA levels for eight DUSPs. In a short inhibitory RNA screen, 12 of 16 DUSPs influenced ERK2 responses. These effects were evident among nuclear inducible MKP, cytoplasmic ERK MKP, JNK/p38 MKP, and atypical DUSP subtypes and, with the exception of the nuclear inducible MKPs, were paralleled by corresponding changes in Egr-1 luciferase activation. Simultaneous removal of all JNK/p38 MKPs or nuclear inducible MKPs revealed them as positive and negative regulators of ERK2 signaling, respectively. The effects of JNK/p38 MKP short inhibitory RNAs were not dependent on protein neosynthesis but were reversed in the presence of JNK and p38 kinase inhibitors, indicating DUSP-mediated cross-talk between MAPK pathways. Overall, our data reveal that a large number of DUSPs influence ERK2 signaling. Together with the known tissue-specific expression of DUSPs and the importance of ERK1/2 in cell regulation, our data support the potential value of DUSPs as targets for drug therapy.
尽管许多刺激可激活细胞外信号调节激酶1和2（ERK1/2），但ERK1/2信号的动力学和区室化取决于刺激，并决定生理后果。ERK可被双特异性磷酸酶（DUSP）失活，特别是丝裂原活化蛋白激酶磷酸酶（MKP）和非典型DUSP，它们既能使ERK1/2去磷酸化，又能作为ERK1/2的支架。我们使用一种细胞成像模型（基于对内源性ERK的敲低以及野生型或突变型ERK2-GFP报告基因的回补），探究了DUSP对瞬时或持续ERK2激活剂（分别为表皮生长因子和佛波醇12,13-二丁酸酯）反应的可能影响。对于这两种刺激，D319N突变（损害DUSP结合）增加了ERK2活性并减少了核积累。这些刺激还增加了8种DUSP的mRNA水平。在一项短发夹RNA筛选中，16种DUSP中的12种影响了ERK2反应。这些效应在核诱导型MKP、细胞质ERK MKP、JNK/p38 MKP和非典型DUSP亚型中很明显，并且除了核诱导型MKP外，Egr-1荧光素酶激活的相应变化与之平行。同时去除所有JNK/p38 MKP或核诱导型MKP后发现，它们分别是ERK2信号传导的正调节因子和负调节因子。JNK/p38 MKP短发夹RNA的作用不依赖于蛋白质的新生合成，但在存在JNK和p38激酶抑制剂的情况下会被逆转，这表明DUSP介导了丝裂原活化蛋白激酶途径之间的串扰。总体而言，我们的数据表明大量DUSP影响ERK2信号传导。结合已知的DUSP组织特异性表达以及ERK1/2在细胞调节中的重要性，我们的数据支持了DUSP作为药物治疗靶点的潜在价值。
