MPF amplification in Xenopus oocyte extracts depends on a two-step activation of cdc25 phosphatase

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.