Bazile Franck, Pascal Aude, Karaiskou Anthi, Chesnel Franck, Kubiak Jacek Z
Biology and Genetics of Development, Mitosis and Meiosis Group, Institute of Genetics and Development of Rennes, University Rennes, Rennes Cedex, France.
Cell Cycle. 2007 Feb 15;6(4):489-96. doi: 10.4161/cc.6.4.3860. Epub 2007 Feb 18.
MPF and MAP kinase ERK2 are two major M-phase kinases. They interact with each other in a complex way during meiotic maturation of Xenopus laevis oocytes. Here we study their interrelationship during first mitosis in X. laevis embryo cell-free extract perturbing the polyubiquitination pathway as a tool. Recombinant ubiquitin K48R (Ub-K48R) mutant protein arrests mitotic cyclin B degradation in the extract. This results in both increased accumulation of phosphorylated form of cyclin B2 and MPF activity as well as mitotic phosphorylation of its substrates. Ub-K48R also increased the mitotic phosphorylation of ERK2. Simultaneous addition of Ub-K48R and the proteasome inhibitor MG 132 strengthened and further prolonged MPF activity, MCM4 phosphorylation and accumulation of phosphorylated forms of cyclin B2. ERK2 phosphorylation levels increased and persisted longer than upon action of Ub-K48R alone. This shows a synergistic effect of inhibition of two different steps of ubiquitin-proteasome pathway on MPF activity and mitotic phosphorylation and ubiquitination of specific M-phase proteins. On the other hand, complete inhibition of ERK2 activation using U0126 had no effect either on MPF activity or on MCM4 phosphorylation either in control or in Ub-K48R-supplemented extracts. Experimental reduction of MPF activity by addition of recombinant p21(Cip) protein resulted in significant reduction of ERK2 phosphorylation. Thus, the reciprocal feedback observed between MPF and ERK2 in meiosis is not observed during mitotic M-phase in cell-free Xenopus embryo extracts. ERK2 phosphorylation is regulated by the levels of MPF activity, however no influence of ERK2 on MPF activity could be detected. These results show a fundamental difference in the relationship between the two major M-phase kinases in meiotic and mitotic cell cycle.
MPF和丝裂原活化蛋白激酶ERK2是两种主要的M期激酶。在非洲爪蟾卵母细胞减数分裂成熟过程中，它们以复杂的方式相互作用。在此，我们以扰乱多聚泛素化途径为工具，研究它们在非洲爪蟾胚胎无细胞提取物第一次有丝分裂过程中的相互关系。重组泛素K48R（Ub-K48R）突变蛋白可阻止提取物中有丝分裂周期蛋白B的降解。这导致周期蛋白B2磷酸化形式的积累增加、MPF活性增强及其底物的有丝分裂磷酸化。Ub-K48R还增加了ERK2的有丝分裂磷酸化。同时添加Ub-K48R和蛋白酶体抑制剂MG 132可增强并进一步延长MPF活性、MCM4磷酸化以及周期蛋白B2磷酸化形式的积累。ERK2磷酸化水平升高且持续时间比单独使用Ub-K48R时更长。这表明抑制泛素-蛋白酶体途径的两个不同步骤对MPF活性、有丝分裂磷酸化以及特定M期蛋白的泛素化具有协同作用）另一方面，在对照提取物或添加Ub-K48R提取物中，使用U0126完全抑制ERK2激活对MPF活性或MCM4磷酸化均无影响）通过添加重组p21（Cip）蛋白实验性降低MPF活性，导致ERK2磷酸化显著降低。因此，在非洲爪蟾胚胎无细胞提取物的有丝分裂M期未观察到减数分裂中MPF和ERK2之间的相互反馈。ERK2磷酸化受MPF活性水平调节，然而未检测到ERK2对MPF活性的影响。这些结果表明减数分裂和有丝分裂细胞周期中两种主要M期激酶之间的关系存在根本差异）
