Control of the pericentrosomal H 2 O 2 level by peroxiredoxin I is critical for mitotic progression

Activation of the Cdk1–cyclin B complex occurs first at the centrosome during prophase, and its amplification through multiple feedback loops involving cyclin B, Cdc25B, Cdc25C, Plk, and Aurora A also occurs at this organelle (Jackman et al., 2003; Bonnet et al., 2008; Lindqvist et al., 2009). Successful cell cycle progression requires that many cell cycle regulators—including cyclins A and B, Plk1, and Aurora A—be degraded in a timely manner. Degradation of these regulators by the 26S proteasome results from their ubiquitination by the multisubunit ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). Activation of APC/C occurs at the centrosome and requires Cdc20 or Cdh1 as an activator protein (Peters, 2006; Pesin and Orr-Weaver, 2008; van Leuken et al., 2008; Wurzenberger and Gerlich, 2011). Cdh1 is prevented from interaction with APC/C when Cdh1 is phosphorylated by Cdks. APC/C–Cdh1 activity thus depends on both Cdks as well as Cdk-opposing phosphatases. The dual-specificity protein tyrosine phosphatase (PTP), Cdc14B, and the Ser/Thr phosphatases, PP1 and PP2A, have been proposed to function as Cdk1-opposing enzymes in mammalian cells (Bassermann et al., 2008; Mochida et al., 2009; Wu et al., 2009; Mocciaro and Schiebel, 2010; Schmitz et al., 2010; Domingo-Sananes et al., 2011). A fraction of each of APC/C, Cdc20, Cdh1, and Cdk1-opposing phosphatases (Cdc14B, PP1, and PP2A) is present at the centrosome (Leach et al., 2003; Cho et al., 2005; Peters, 2006; Wu et al., 2008; Schmitz et al., 2010), as are Cdk1, Cdc25, cyclin B, Plk1, and Aurora A. At the onset of mitosis, Cdk1–cyclin B activity begins to increase as a result of positive feedback loops including cyclin B, Cdc25, Plk1, and Aurora A. The low level of incipient Cdk1 activity is likely insufficient to allow the accumulation of phosphorylated Cdh1 at the centrosome in the absence of concurrent suppression of the activity of Cdk1-opposing phosphatases, which, together with Cdh1, are enriched at this organelle. In the absence of such suppression of centrosomal phosphatase activity, further activation of Cdk1 would not be expected to occur because of the premature degradation of cyclin B, Plk1, and Aurora A. Here, we find that the centrosomal levels of cyclin B, Plk1, and Aurora A as well as mitotic entry are likely regulated by the local concentration of H2O2 around the centrosome. We were led into this study by our previous observation that PrxI is inactivated when phosphorylated on Thr by purified Cdk1–cyclin B (Chang et al., 2002). Peroxiredoxins (Prxs) are a major class of H2O2-eliminating enzymes (Rhee et al., 2012). MamProteins associated with the centrosome play key roles in mitotic progression in mammalian cells. The activity of Cdk1-opposing phosphatases at the centrosome must be inhibited during early mitosis to prevent premature dephosphorylation of Cdh1—an activator of the ubiquitin ligase anaphase-promoting complex/cyclosome—and the consequent premature degradation of mitotic activators. In this paper, we show that reversible oxidative inactivation of centrosome-bound protein phosphatases such as Cdc14B by H2O2 is likely responsible for this inhibition. The intracellular concentration of H2O2 increases as the cell cycle progresses. Whereas the centrosome is shielded from H2O2 through its association with the H2O2-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H2O2 and facilitating inactivation of centrosome-bound phosphatases. Dephosphorylation of PrxI by okadaic acid–sensitive phosphatases during late mitosis again shields the centrosome from H2O2 and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle. Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression