Karaïskou A, Cayla X, Haccard O, Jessus C, Ozon R
Laboratoire de Physiologie de la Reproduction, INRA/URA-CNRS 1449, Université Pierre et Marie Curie, Boîte 13, 4 place Jussieu, Paris cedex 05, 75252, France.
Exp Cell Res. 1998 Nov 1;244(2):491-500. doi: 10.1006/excr.1998.4220.
The activation of Cdc2 kinase induces the entry into M-phase of all eukaryotic cells. We have developed a cell-free system prepared from prophase-arrested Xenopus oocytes to analyze the mechanism initiating the all-or-none activation of Cdc2 kinase. Inhibition of phosphatase 2A, the major okadaic acid-sensitive Ser/Thr phosphatase, in these extracts, provokes Cdc2 kinase amplification and concomitant hyperphosphorylation of Cdc25 phosphatase, with a lag of about 1 h. Polo-like kinase (Plx1 kinase) is activated slightly after Cdc2. All these events are totally inhibited by the cdk inhibitor p21(Cip1), demonstrating that Plx1 kinase activation depends on Cdc2 kinase activity. Addition of a threshold level of recombinant Cdc25 induces a linear activation of Cdc2 and Plx1 kinases and a partial phosphorylation of Cdc25. We propose that the Cdc2 positive feedback loop involves two successive phosphorylation steps of Cdc25 phosphatase: the first one is catalyzed by Cdc2 kinase and/or Plx1 kinase but it does not modify Cdc25 enzymatic activity, the second one requires a new kinase counteracted by phosphatase 2A. Furthermore we demonstrate that, under our conditions, Cdc2 amplification and MAP kinase activation are two independent events.
Cdc2激酶的激活促使所有真核细胞进入M期。我们开发了一种由处于前期阻滞的非洲爪蟾卵母细胞制备的无细胞系统，以分析引发Cdc2激酶全或无激活的机制。在这些提取物中，抑制磷酸酶2A（主要的冈田酸敏感型丝氨酸/苏氨酸磷酸酶）会引发Cdc2激酶的扩增以及Cdc25磷酸酶的伴随性过度磷酸化，有大约1小时的延迟。Polo样激酶（Plx1激酶）在Cdc2之后稍有激活。所有这些事件都被细胞周期蛋白依赖性激酶抑制剂p21（Cip1）完全抑制，表明Plx1激酶的激活依赖于Cdc2激酶的活性。添加阈值水平的重组Cdc25会诱导Cdc2和Plx1激酶的线性激活以及Cdc25的部分磷酸化。我们提出，Cdc2正反馈环涉及Cdc25磷酸酶的两个连续磷酸化步骤：第一个由Cdc2激酶和/或Plx1激酶催化，但不改变Cdc25的酶活性，第二个需要一种新的激酶，该激酶会被磷酸酶2A抵消。此外，我们证明，在我们的条件下，Cdc2扩增和丝裂原活化蛋白激酶激活是两个独立的事件。
