Published July 6, 2015 This article has original data in the JCB Data Viewer JCB: Report http://jcb-dataviewer.rupress.org/jcb/browse/10902 Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression Jung Mi Lim,1 Kyung S. Lee,2 Hyun Ae Woo,1 Dongmin Kang,1 and Sue Goo Rhee3 1Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, South Korea 2Laboratory of Metabolism, National Cancer Institute, Bethesda, MD 20892 3Yonsei Biomedical Research Institute, Yonsei University, Seoul 120-749, South Korea Proteins associated with the centrosome play key roles in mitotic progression in mammalian cells. The activity of Cdk1-op- posing 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 in- activated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H2O2 and facili- Downloaded from tating 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. Introduction on April 13, 2017 Activation of the Cdk1–cyclin B complex occurs first at the cen- et al., 2009; Mocciaro and Schiebel, 2010; Schmitz et al., 2010; trosome during prophase, and its amplification through multiple Domingo-Sananes et al., 2011). feedback loops involving cyclin B, Cdc25B, Cdc25C, Plk, and A fraction of each of APC/C, Cdc20, Cdh1, and Cdk1-op- Aurora A also occurs at this organelle (Jackman et al., 2003; posing phosphatases (Cdc14B, PP1, and PP2A) is present at the Bonnet et al., 2008; Lindqvist et al., 2009). Successful cell cycle centrosome (Leach et al., 2003; Cho et al., 2005; Peters, 2006; progression requires that many cell cycle regulators—including Wu et al., 2008; Schmitz et al., 2010), as are Cdk1, Cdc25, cyclin cyclins A and B, Plk1, and Aurora A—be degraded in a timely B, Plk1, and Aurora A. At the onset of mitosis, Cdk1–cyclin B manner. Degradation of these regulators by the 26S proteasome activity begins to increase as a result of positive feedback loops results from their ubiquitination by the multisubunit ubiqui- including cyclin B, Cdc25, Plk1, and Aurora A. The low level tin ligase anaphase-promoting complex/cyclosome (APC/C). of incipient Cdk1 activity is likely insufficient to allow the ac- Activation of APC/C occurs at the centrosome and requires cumulation of phosphorylated Cdh1 at the centrosome in the THE JOURNAL OF CELL BIOLOGY Cdc20 or Cdh1 as an activator protein (Peters, 2006; Pesin and absence of concurrent suppression of the activity of Cdk1-op- Orr-Weaver, 2008; van Leuken et al., 2008; Wurzenberger and posing phosphatases, which, together with Cdh1, are enriched Gerlich, 2011). Cdh1 is prevented from interaction with APC/C at this organelle. In the absence of such suppression of centro- when Cdh1 is phosphorylated by Cdks. APC/C–Cdh1 activity somal phosphatase activity, further activation of Cdk1 would thus depends on both Cdks as well as Cdk-opposing phospha- not be expected to occur because of the premature degradation tases. The dual-specificity protein tyrosine phosphatase (PTP), of cyclin B, Plk1, and Aurora A. Cdc14B, and the Ser/Thr phosphatases, PP1 and PP2A, have Here, we find that the centrosomal levels of cyclin B, been proposed to function as Cdk1-opposing enzymes in mam- Plk1, and Aurora A as well as mitotic entry are likely regulated malian cells (Bassermann et al., 2008; Mochida et al., 2009; Wu 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 Thr90 by purified Cdk1–cy- clin B (Chang et al., 2002). Peroxiredoxins (Prxs) are a major Correspondence to Dongmin Kang: [email protected]; or Sue Goo Rhee: [email protected] class of H2O2-eliminating enzymes (Rhee et al., 2012). Mam- Abbreviations used in this paper: APC/C, anaphase-promoting complex/ cyclosome; DPI, diphenyleneiodonium; MAFP, methyl arachidonyl fluorophos- © 2015 Lim et al. This article is distributed under the terms of an Attribution–Noncommercial– phonate; MEF, mouse embryonic fibroblast; NDGA, nordihydroguaiaretic acid; Share Alike–No Mirror Sites license for the first six months after the publication date (see NEM, N-ethylmaleimide; OA, okadaic acid; PACT, pericentrin-AKAP450 cen- http://www.rupress.org/terms). After six months it is available under a Creative Commons trosomal targeting; pHH3, phosphorylated histone H3; Prx, peroxiredoxin; PTP, License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at protein tyrosine phosphatase; SIM, structured illumination microscopy. http://creativecommons.org/licenses/by-nc-sa/3.0/). Supplemental Material can be found at: /content/suppl/2015/07/06/jcb.201412068.DC1.html Original image data can be found at: The Rockefeller University Press $30.00 http://jcb-dataviewer.rupress.org/jcb/browse/10902 J. Cell Biol. Vol. 210 No. 1 23–33 www.jcb.org/cgi/doi/10.1083/jcb.201412068 JCB 23 Published July 6, 2015 malian cells express six Prx isoforms (PrxI to PrxVI), which are with a retroviral vector encoding a modified form of catalase implicated in a variety of cellular processes. either with a centrosome-targeting pericentrin-AKAP450 cen- trosomal targeting (PACT) sequence (Cat-PACT) or with the corresponding sequence lacking the centrosome-targeting core Results and discussion (Cat-delPACT; Fig. 2 A; Gillingham and Munro, 2000). The presence of Cat-PACT and the absence of Cat-delPACT at the Phosphorylation of centrosome-associated centrosome were verified by confocal immunofluorescence mi- PrxI in early mitotic cells croscopy (Fig. 2 B). HeLa cells that had been infected with the Whereas high H2O2 levels induce cell cycle arrest, low H2O2 empty, Cat-PACT, or Cat-delPACT vectors were synchronized levels are required for G1–S and G2–M phase transitions (Ha- at G1–S by thymidine treatment and released in the presence vens et al., 2006; Yamaura et al., 2009). The molecular mech- of nocodazole for 10 h (Fig. 2 C). Early mitotic cells were anisms by which H2O2 modulates cell cycle progression have scored on the basis of cell rounding (Fig. 2 D) and chromosome remained unclear, however. To examine the possible link be- condensation (Fig. 2 E). The number of mitotic cells (mean of tween the role of H2O2 in cell cycle regulation and PrxI phos- values from Fig. 2, D and E) for cells expressing Cat-PACT 90 phorylation on Thr , we monitored this latter event during was reduced by ∼45% and ∼35% compared with that for cells the cell cycle in HeLa cells that had been synchronized at the infected with the empty vector or the vector encoding Cat-del- G1–S border (0 h) with a double thymidine block and then re- PACT, respectively. Immunoblot analysis of the infected HeLa leased for various times. Phosphorylated PrxI (pPrxI) appeared cells showed that the level of inactive (Tyr15 phosphorylated) slightly earlier than did the mitotic marker phosphorylated Cdk1 in Cat-PACT cells was about twice that in Cat-delPACT histone H3 (pHH3), and it disappeared in parallel with pHH3 cells and that the level of pHH3 in Cat-PACT cells was ∼50% of (Fig. 1 A). When HeLa or U2OS cells arrested in prometaphase that in Cat-delPACT cells (Fig. 2, F and G). Such analysis also with nocodazole were released from the arrest, pPrxI disap- revealed that Cat-PACT and Cat-delPACT were expressed sta- peared rapidly, with the rate of its loss being slightly greater bly at low levels relative to endogenous peroxisomal catalase, than that for cyclin B1 or pHH3 (Fig. S1 A). suggesting that any effect of Cat-PACT present in the cytosol Downloaded from The amount of pPrxI in prometaphase HeLa cells was es- instead of at the centrosome as a result of excessive expression timated to be ∼0.4% of total PrxI (Fig. S1 B). We therefore rea- was minimal (Fig. 2 F). We also monitored mitotic entry by soned that PrxI phosphorylation is likely a localized event, and fluorescence imaging of live cells after the release of Cat-PACT we searched for its location in asynchronously growing HeLa and Cat-delPACT cells from G1–S arrest. Mitotic entry of Cat- cells using confocal microscopy. pPrxI was found to colocalize PACT cells was delayed by a mean of ∼2–3 h compared with with the centrosome marker γ-tubulin at early stages of mitosis that of Cat-delPACT cells (Fig. 2 H; and Videos 1 and 2). These (prometaphase and metaphase) but not during interphase or late results thus supported the notion that exposure of the centro- on April 13, 2017 mitotic stages (anaphase, telophase, and cytokinesis; Fig. 1 B). some to H2O2 is required for normal mitotic entry. The centrosome is composed of a pair of centrioles surrounded We also found that the centrosomal levels of cyclin B1, by pericentriolar material. Structured illumination microscopy Plk1, and Aurora A in Cat-PACT cells were reduced by 55, 30, (SIM; Fig. 1 C) revealed that although pPrxI is colocalized with and 40%, respectively, compared with those in Cat-delPACT Cep192, a pericentriolar material marker of mitotic cells (Kim cells, suggesting that exposure of the centrosome to H2O2 is et al., 2013), there are also regions of Cep192 that do not ex- likely required to protect them from degradation (Fig.