Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression.
Bottom Line: 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.
Affiliation: Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, South Korea.Show MeSH
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Mentions: To examine the effect of exposure of the centrosome to cytoplasmic H2O2, which would be expected to occur as a result of PrxI phosphorylation (inactivation), we infected HeLa cells with a retroviral vector encoding a modified form of catalase either with a centrosome-targeting pericentrin-AKAP450 centrosomal targeting (PACT) sequence (Cat-PACT) or with the corresponding sequence lacking the centrosome-targeting core (Cat-delPACT; Fig. 2 A; Gillingham and Munro, 2000). The presence of Cat-PACT and the absence of Cat-delPACT at the centrosome were verified by confocal immunofluorescence microscopy (Fig. 2 B). HeLa cells that had been infected with the empty, Cat-PACT, or Cat-delPACT vectors were synchronized at G1–S by thymidine treatment and released in the presence of nocodazole for 10 h (Fig. 2 C). Early mitotic cells were scored on the basis of cell rounding (Fig. 2 D) and chromosome condensation (Fig. 2 E). The number of mitotic cells (mean of values from Fig. 2, D and E) for cells expressing Cat-PACT was reduced by ∼45% and ∼35% compared with that for cells infected with the empty vector or the vector encoding Cat-delPACT, respectively. Immunoblot analysis of the infected HeLa cells showed that the level of inactive (Tyr15 phosphorylated) Cdk1 in Cat-PACT cells was about twice that in Cat-delPACT cells and that the level of pHH3 in Cat-PACT cells was ∼50% of that in Cat-delPACT cells (Fig. 2, F and G). Such analysis also revealed that Cat-PACT and Cat-delPACT were expressed stably at low levels relative to endogenous peroxisomal catalase, suggesting that any effect of Cat-PACT present in the cytosol instead of at the centrosome as a result of excessive expression was minimal (Fig. 2 F). We also monitored mitotic entry by fluorescence imaging of live cells after the release of Cat-PACT and Cat-delPACT cells from G1–S arrest. Mitotic entry of Cat-PACT cells was delayed by a mean of ∼2–3 h compared with that of Cat-delPACT cells (Fig. 2 H; and Videos 1 and 2). These results thus supported the notion that exposure of the centrosome to H2O2 is required for normal mitotic entry.
Affiliation: Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, South Korea.