Aurora-A kinase and inhibitor-2 regulate the cyclin threshold for mitotic entry in Xenopus early embryonic cell cycles.
The abrupt activation of CDK1 during mitotic entry requires suppression of CDK activity until a threshold concentration of cyclin B is synthesized, triggering the activation of a large pool of CDK. The cellular mechanisms that define the concentration of cyclin B at which the threshold occurs are unknown. Here we demonstrate that this threshold is regulated by Aurora-A kinase and phosphatase Inhibitor-2. In Xenopus CSF extracts that actively translate cyclin B1, immunodepletion of either endogenous xInhibitor-2 or endogenous xAurora-A caused delayed mitotic entry and normal timing was restored by addition of the respective recombinant proteins. Aurora-A depleted extracts also could be rescued by the addition of full-length xInhibitor-2, but not an xInhibitor-2 truncated of its PP1 binding motif. This demonstrates that inhibition of PP1 was required to compensate for the absence of Aurora-A. To test the hypothesis that the delays in mitotic entry in CSF extracts were due to increases in cyclin B thresholds, we employed interphase extracts, which are driven into mitosis by the addition of recombinant cyclin B in a nonlinear (threshold) dose-response. Neutralization of endogenous xInhibitor-2 or xAurora with antibodies increased the cyclin B threshold concentration. Alternatively, the addition of exogenous Aurora-A or Inhibitor-2 lowered the concentration of cyclin B that triggered CDK activation. Because the cyclin B threshold could be raised or lowered by changing the amount of either Aurora-A or Inhibitor-2, the results demonstrate these regulatory proteins are involved in a signaling loop required to create the switching behavior characteristic of mitotic entry.
Satinover
David L
DL
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Brautigan
David L
DL
Stukenberg
P Todd
PT
D000818:Animals; D064127:Aurora Kinases; D002453:Cell Cycle; D019926:Cyclin B / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004625:Embryo, Nonmammalian; D008938:Mitosis; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000494:pharmacology / Q000502:physiology*; D011506:Proteins / Q000494:pharmacology / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D014981:Xenopus; D029867:Xenopus Proteins / Q000502:physiology*; D000097847:Protein Phosphatase Inhibitory Proteins
D064127:Aurora Kinases; D019926:Cyclin B; D018844:Cyclin-Dependent Kinases; D010749:Phosphoprotein Phosphatases; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins