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Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression.

Lim JM, Lee KS, Woo HA, Kang D, Rhee SG - J. Cell Biol. (2015)

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, South Korea.

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Phosphorylation of PrxI at Thr90 occurs at the centrosome of HeLa cells during early mitosis. (A, top) HeLa cells that had been arrested at the G1–S border with a double thymidine block (T/T) were released in fresh medium (at 0 h) and collected at the indicated times for immunoblot analysis with antibodies to the indicated proteins. (bottom) The percentage of cells in the various phases of the cell cycle was estimated by flow cytometric analysis. Data are representative of three experiments with similar results. (B) Confocal microscopy of asynchronously growing HeLa cells stained with antibodies to pPrxI (green) and to γ-tubulin (red). Cell cycle stage was monitored by staining of DNA with DAPI (blue) and bright-field imaging. The areas indicated by the arrows are shown at higher magnification in the insets. (C) 3D-SIM images of pPrxI (green) on two pericentrosomes (C1 and C2) of mitotic HeLa cells. Fluorescent line intensity histograms show the colocalization of pPrxI (green) with pericentrosomal protein Cep192 (red) in two centrosomes. 3D-rendering image is shown in one centrosome. The data shown are from a single representative experiment out of three repeats. Arrows indicate pericentrosomes. (D) HeLa cells synchronized at prometaphase by treatment with thymidine and nocodazole (synchronous [Syn]) or those growing asynchronously (Asyn) were subjected to subcellular fractionation. Total cell lysates without nuclei (Tot) as well as cytosolic (Cyt) and centrosome (Cen) fractions were subjected to immunoblot analysis with antibodies to the indicated proteins.
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fig1: Phosphorylation of PrxI at Thr90 occurs at the centrosome of HeLa cells during early mitosis. (A, top) HeLa cells that had been arrested at the G1–S border with a double thymidine block (T/T) were released in fresh medium (at 0 h) and collected at the indicated times for immunoblot analysis with antibodies to the indicated proteins. (bottom) The percentage of cells in the various phases of the cell cycle was estimated by flow cytometric analysis. Data are representative of three experiments with similar results. (B) Confocal microscopy of asynchronously growing HeLa cells stained with antibodies to pPrxI (green) and to γ-tubulin (red). Cell cycle stage was monitored by staining of DNA with DAPI (blue) and bright-field imaging. The areas indicated by the arrows are shown at higher magnification in the insets. (C) 3D-SIM images of pPrxI (green) on two pericentrosomes (C1 and C2) of mitotic HeLa cells. Fluorescent line intensity histograms show the colocalization of pPrxI (green) with pericentrosomal protein Cep192 (red) in two centrosomes. 3D-rendering image is shown in one centrosome. The data shown are from a single representative experiment out of three repeats. Arrows indicate pericentrosomes. (D) HeLa cells synchronized at prometaphase by treatment with thymidine and nocodazole (synchronous [Syn]) or those growing asynchronously (Asyn) were subjected to subcellular fractionation. Total cell lysates without nuclei (Tot) as well as cytosolic (Cyt) and centrosome (Cen) fractions were subjected to immunoblot analysis with antibodies to the indicated proteins.

Mentions: Whereas high H2O2 levels induce cell cycle arrest, low H2O2 levels are required for G1–S and G2–M phase transitions (Havens et al., 2006; Yamaura et al., 2009). The molecular mechanisms by which H2O2 modulates cell cycle progression have remained unclear, however. To examine the possible link between the role of H2O2 in cell cycle regulation and PrxI phosphorylation on Thr90, we monitored this latter event during the cell cycle in HeLa cells that had been synchronized at the G1–S border (0 h) with a double thymidine block and then released for various times. Phosphorylated PrxI (pPrxI) appeared slightly earlier than did the mitotic marker phosphorylated histone H3 (pHH3), and it disappeared in parallel with pHH3 (Fig. 1 A). When HeLa or U2OS cells arrested in prometaphase with nocodazole were released from the arrest, pPrxI disappeared rapidly, with the rate of its loss being slightly greater than that for cyclin B1 or pHH3 (Fig. S1 A).


Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression.

Lim JM, Lee KS, Woo HA, Kang D, Rhee SG - J. Cell Biol. (2015)

Phosphorylation of PrxI at Thr90 occurs at the centrosome of HeLa cells during early mitosis. (A, top) HeLa cells that had been arrested at the G1–S border with a double thymidine block (T/T) were released in fresh medium (at 0 h) and collected at the indicated times for immunoblot analysis with antibodies to the indicated proteins. (bottom) The percentage of cells in the various phases of the cell cycle was estimated by flow cytometric analysis. Data are representative of three experiments with similar results. (B) Confocal microscopy of asynchronously growing HeLa cells stained with antibodies to pPrxI (green) and to γ-tubulin (red). Cell cycle stage was monitored by staining of DNA with DAPI (blue) and bright-field imaging. The areas indicated by the arrows are shown at higher magnification in the insets. (C) 3D-SIM images of pPrxI (green) on two pericentrosomes (C1 and C2) of mitotic HeLa cells. Fluorescent line intensity histograms show the colocalization of pPrxI (green) with pericentrosomal protein Cep192 (red) in two centrosomes. 3D-rendering image is shown in one centrosome. The data shown are from a single representative experiment out of three repeats. Arrows indicate pericentrosomes. (D) HeLa cells synchronized at prometaphase by treatment with thymidine and nocodazole (synchronous [Syn]) or those growing asynchronously (Asyn) were subjected to subcellular fractionation. Total cell lysates without nuclei (Tot) as well as cytosolic (Cyt) and centrosome (Cen) fractions were subjected to immunoblot analysis with antibodies to the indicated proteins.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig1: Phosphorylation of PrxI at Thr90 occurs at the centrosome of HeLa cells during early mitosis. (A, top) HeLa cells that had been arrested at the G1–S border with a double thymidine block (T/T) were released in fresh medium (at 0 h) and collected at the indicated times for immunoblot analysis with antibodies to the indicated proteins. (bottom) The percentage of cells in the various phases of the cell cycle was estimated by flow cytometric analysis. Data are representative of three experiments with similar results. (B) Confocal microscopy of asynchronously growing HeLa cells stained with antibodies to pPrxI (green) and to γ-tubulin (red). Cell cycle stage was monitored by staining of DNA with DAPI (blue) and bright-field imaging. The areas indicated by the arrows are shown at higher magnification in the insets. (C) 3D-SIM images of pPrxI (green) on two pericentrosomes (C1 and C2) of mitotic HeLa cells. Fluorescent line intensity histograms show the colocalization of pPrxI (green) with pericentrosomal protein Cep192 (red) in two centrosomes. 3D-rendering image is shown in one centrosome. The data shown are from a single representative experiment out of three repeats. Arrows indicate pericentrosomes. (D) HeLa cells synchronized at prometaphase by treatment with thymidine and nocodazole (synchronous [Syn]) or those growing asynchronously (Asyn) were subjected to subcellular fractionation. Total cell lysates without nuclei (Tot) as well as cytosolic (Cyt) and centrosome (Cen) fractions were subjected to immunoblot analysis with antibodies to the indicated proteins.
Mentions: Whereas high H2O2 levels induce cell cycle arrest, low H2O2 levels are required for G1–S and G2–M phase transitions (Havens et al., 2006; Yamaura et al., 2009). The molecular mechanisms by which H2O2 modulates cell cycle progression have remained unclear, however. To examine the possible link between the role of H2O2 in cell cycle regulation and PrxI phosphorylation on Thr90, we monitored this latter event during the cell cycle in HeLa cells that had been synchronized at the G1–S border (0 h) with a double thymidine block and then released for various times. Phosphorylated PrxI (pPrxI) appeared slightly earlier than did the mitotic marker phosphorylated histone H3 (pHH3), and it disappeared in parallel with pHH3 (Fig. 1 A). When HeLa or U2OS cells arrested in prometaphase with nocodazole were released from the arrest, pPrxI disappeared rapidly, with the rate of its loss being slightly greater than that for cyclin B1 or pHH3 (Fig. S1 A).

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, South Korea.

Show MeSH
Related in: MedlinePlus