Mody N, Leitch J, Armstrong C, Dixon J, Cohen P
Medical Research Council Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK.
FEBS Lett. 2001 Jul 27;502(1-2):21-4. doi: 10.1016/s0014-5793(01)02651-5.
Antibodies that recognise the active phosphorylated forms of mitogen-activated protein kinase (MAPK) kinase 5 (MKK5) and extracellular signal-regulated kinase 5 (ERK5) in untransfected cells have been exploited to show that the epidermal growth factor (EGF)-induced activation of MKK5 and ERK5 occurs subsequent to the activation of ERK1 and ERK2 in HeLa cells. The drugs U0126 and PD184352, which prevent the activation of MKK1 (and hence the activation of ERK1/ERK2), also prevent the activation of MKK5, although higher concentrations are required. Our studies define physiological targets of the MKK5/ERK5 pathway as proteins whose phosphorylation is largely prevented by 10 microM PD184352, but unaffected by 2 microM PD184352. Surprisingly, 2 microM PD184352 prolongs the activation of MKK5 and ERK5 induced by EGF or H(2)O(2), indicating negative control of the MKK5/ERK5 pathway by the classical MAPK cascade. Our results also indicate that ERK5 is not a significant activator of MAPK-activated protein kinase-1/RSK in HeLa cells.
能够识别未转染细胞中有活性的磷酸化丝裂原活化蛋白激酶（MAPK）激酶5（MKK5）和细胞外信号调节激酶5（ERK5）的抗体已被用于表明，在HeLa细胞中，表皮生长因子（EGF）诱导的MKK5和ERK5激活发生在ERK1和ERK2激活之后。药物U0126和PD184352可阻止MKK1的激活（从而阻止ERK1/ERK2的激活），尽管需要更高的浓度，但它们也能阻止MKK5的激活。我们的研究将MKK5/ERK5途径的生理靶点定义为那些磷酸化在很大程度上被10微摩尔PD184352阻止，但不受2微摩尔PD184352影响的蛋白质。令人惊讶的是，2微摩尔PD184352会延长由EGF或H₂O₂诱导的MKK5和ERK5的激活，这表明经典的MAPK级联对MKK5/ERK5途径有负调控作用。我们的结果还表明，在HeLa细胞中，ERK5不是MAPK激活的蛋白激酶-1/RSK的重要激活剂。
