Katsu Y, Minshall N, Nagahama Y, Standart N
Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, England.
Dev Biol. 1999 May 1;209(1):186-99. doi: 10.1006/dbio.1999.9247.
During early development gene expression is controlled principally at the translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs which are translationally dormant or masked until meiotic maturation. Fertilisation of the oocyte leads to rapid polysomal recruitment of the abundant cyclin and ribonucleotide reductase mRNAs at about the time they undergo cytoplasmic polyadenylation. Clam p82, a 3' UTR RNA-binding protein, and a member of the CPEB (cytoplasmic polyadenylation element binding protein) family, functions as a translational masking factor in oocytes and as a polyadenylation factor in fertilised eggs. In meiotically maturing clam oocytes, p82/CPEB is rapidly phosphorylated on multiple residues to a 92-kDa apparent size, prior to its degradation during the first cell cleavage. Here we examine the protein kinase(s) that phosphorylates clam p82/CPEB using a clam oocyte activation cell-free system that responds to elevated pH, mirroring the pH rise that accompanies fertilisation. We show that p82/CPEB phosphorylation requires Ca2+ (<100 microM) in addition to raised pH. Examination of the calcium dependency combined with the use of specific inhibitors implicates the combined and independent actions of cdc2 and MAP kinases in p82/CPEB phosphorylation. Calcium is necessary for both the activation and the maintenance of MAP kinase, whose activity is transient in vitro, as in vivo. While cdc2 kinase plays a role in the maintenance of MAP kinase activity, it is not required for the activation of MAP kinase. We propose a model of clam p82/CPEB phosphorylation in which MAP kinase initially phosphorylates clam p82/CPEB, at a minor subset of sites that does not alter its migration, and cdc2 kinase is necessary for the second wave of phosphorylation that results in the large mobility size shift of clam p82/CPEB. The possible roles of phosphorylation for the function and regulation of p82/CPEB are discussed.
在早期发育过程中，基因表达主要在翻译水平受到调控。硬壳蛤（Spisula solidissima）的卵母细胞含有大量母源mRNA储备，这些mRNA在减数分裂成熟之前处于翻译休眠或被掩盖状态。卵母细胞受精后，在丰富的细胞周期蛋白和核糖核苷酸还原酶mRNA进行细胞质多聚腺苷酸化时，它们会迅速被募集到多核糖体上。蛤p82是一种3'UTR RNA结合蛋白，属于CPEB（细胞质多聚腺苷酸化元件结合蛋白）家族成员，在卵母细胞中作为翻译掩盖因子，在受精卵中作为多聚腺苷酸化因子发挥作用。在减数分裂成熟的蛤卵母细胞中，p82/CPEB在多个位点迅速磷酸化，表观大小变为92 kDa，然后在第一次细胞分裂期间降解。在这里，我们使用一种蛤卵母细胞激活无细胞系统来研究磷酸化蛤p82/CPEB的蛋白激酶，该系统对pH升高有反应，模拟受精时伴随的pH升高。我们发现，除了pH升高外，p82/CPEB磷酸化还需要Ca2+（<100 microM）。对钙依赖性的研究以及使用特异性抑制剂表明，cdc2和MAP激酶在p82/CPEB磷酸化过程中具有联合和独立的作用。钙对于MAP激酶的激活和维持都是必需的，其活性在体外和体内一样是短暂的。虽然cdc2激酶在维持MAP激酶活性方面发挥作用，但它不是MAP激酶激活所必需的。我们提出了一个蛤p82/CPEB磷酸化模型，其中MAP激酶最初在不改变其迁移率的少数位点磷酸化蛤p82/CPEB，而cdc2激酶对于导致蛤p82/CPEB迁移率大幅变化的第二波磷酸化是必需的。我们还讨论了磷酸化对p82/CPEB功能和调控的可能作用。
