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The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification.

Brownlee CW, Klebba JE, Buster DW, Rogers GC - J. Cell Biol. (2011)

Bottom Line: However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown.However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification.We demonstrate that ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA.

ABSTRACT
Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. In this paper, we show that PP2A (Protein Phosphatase 2A(Twins)) counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. We demonstrate that ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification.

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PP2ATws is required for centriole duplication. (A) 5-d RNAi-treated or 24-h OA-treated S2 cells were immunostained for PLP to mark centrioles and Hoechst stained to label DNA. Cell borders are traced with dashed lines. Bar, 5 µm. (B) PP2A inhibition by Mts, 29B, or Tws RNAi leads to centriole loss. After RNAi treatment, the number of PLP-immunostained centrioles per cell was measured. Graph shows the percentage of cells with the indicated number of centrioles per cell. Each number in a bar is the percent mean for two experiments (n = 500 cells/treatment). Asterisks mark significant differences (relative to control) for comparisons mentioned in the text (P < 0.02). Error bars indicate SD. (C) Mts cosediments with centrioles purified from mitotic S2 cells on a 20–70% sucrose gradient. Fractions (numbered) were immunoblotted for the indicated proteins. Asterisks mark the major centriole-containing fractions. (D) Tws protein is maximal during mitosis. (left) Graph of normalized endogenous Mts and Tws levels in asynchronous cells (Asynch) or after a 24-h drug-induced cell cycle arrest. Plotted values were determined from the anti-Mts and Tws immunoblots (right) shown. The graph and blots are representative examples of three independent experiments, all with similar results. α-Tubulin (α Tub) was used as a loading control. Wdb, Widerborst; Wrd, Well rounded.
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fig2: PP2ATws is required for centriole duplication. (A) 5-d RNAi-treated or 24-h OA-treated S2 cells were immunostained for PLP to mark centrioles and Hoechst stained to label DNA. Cell borders are traced with dashed lines. Bar, 5 µm. (B) PP2A inhibition by Mts, 29B, or Tws RNAi leads to centriole loss. After RNAi treatment, the number of PLP-immunostained centrioles per cell was measured. Graph shows the percentage of cells with the indicated number of centrioles per cell. Each number in a bar is the percent mean for two experiments (n = 500 cells/treatment). Asterisks mark significant differences (relative to control) for comparisons mentioned in the text (P < 0.02). Error bars indicate SD. (C) Mts cosediments with centrioles purified from mitotic S2 cells on a 20–70% sucrose gradient. Fractions (numbered) were immunoblotted for the indicated proteins. Asterisks mark the major centriole-containing fractions. (D) Tws protein is maximal during mitosis. (left) Graph of normalized endogenous Mts and Tws levels in asynchronous cells (Asynch) or after a 24-h drug-induced cell cycle arrest. Plotted values were determined from the anti-Mts and Tws immunoblots (right) shown. The graph and blots are representative examples of three independent experiments, all with similar results. α-Tubulin (α Tub) was used as a loading control. Wdb, Widerborst; Wrd, Well rounded.

Mentions: PP2A is a holoenzyme composed of three subunits: a catalytic C subunit (microtubule star [Mts] in Drosophila), a structural A subunit (Drosophila PP2A-29B), and a regulatory B subunit encoded by one of four fly genes (Tws/PR55, Widerborst, Well rounded, or PR72; Mayer-Jaekel et al., 1992; Uemura et al., 1993; Snaith et al., 1996; Hannus et al., 2002; Li et al., 2002; Viquez et al., 2006). To test whether PP2A is involved in centriole duplication, we used RNAi to deplete each protein individually (when antibodies were available, we confirmed depletion by immunoblotting; Fig. S3 A) and measured centriole numbers in S2 cells. Depletion of Mts or PP2A-29B resulted in loss of centrioles (Fig. 2 A), significantly increasing the percentage of cells with less than two centrioles compared with controls (Fig. 2 B). This effect was phenocopied by treating cells with the PP2A inhibitor okadaic acid (OA; Fig. 2, A and B). Depletion of only one regulatory subunit, Tws, also significantly decreased centriole number (Fig. 2 B). Prolonged Tws RNAi had no effect on cell cycle progression (Fig. S3 B) or mitotic index (control RNAi = 2.0%; Tws RNAi = 2.7%) but dramatically elevated the frequency of mitotic spindles lacking centrosomes (similar experiments with prolonged Mts or PP2A-29B RNAi led to significant cell death; Fig. S3 C). Consistent with a role in centriole duplication, Mts and PP2A-29B localize to mitotic centrioles in S2R+ cells (Dobbelaere et al., 2008). Likewise, we found a fraction of Mts copurified with centrioles isolated from mitotic S2 cells using sucrose gradient centrifugation (Fig. 2 C). Together, these results suggest that a PP2ATws complex is required for centriole duplication and is appropriately positioned on mitotic centrioles.


The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification.

Brownlee CW, Klebba JE, Buster DW, Rogers GC - J. Cell Biol. (2011)

PP2ATws is required for centriole duplication. (A) 5-d RNAi-treated or 24-h OA-treated S2 cells were immunostained for PLP to mark centrioles and Hoechst stained to label DNA. Cell borders are traced with dashed lines. Bar, 5 µm. (B) PP2A inhibition by Mts, 29B, or Tws RNAi leads to centriole loss. After RNAi treatment, the number of PLP-immunostained centrioles per cell was measured. Graph shows the percentage of cells with the indicated number of centrioles per cell. Each number in a bar is the percent mean for two experiments (n = 500 cells/treatment). Asterisks mark significant differences (relative to control) for comparisons mentioned in the text (P < 0.02). Error bars indicate SD. (C) Mts cosediments with centrioles purified from mitotic S2 cells on a 20–70% sucrose gradient. Fractions (numbered) were immunoblotted for the indicated proteins. Asterisks mark the major centriole-containing fractions. (D) Tws protein is maximal during mitosis. (left) Graph of normalized endogenous Mts and Tws levels in asynchronous cells (Asynch) or after a 24-h drug-induced cell cycle arrest. Plotted values were determined from the anti-Mts and Tws immunoblots (right) shown. The graph and blots are representative examples of three independent experiments, all with similar results. α-Tubulin (α Tub) was used as a loading control. Wdb, Widerborst; Wrd, Well rounded.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3198173&req=5

fig2: PP2ATws is required for centriole duplication. (A) 5-d RNAi-treated or 24-h OA-treated S2 cells were immunostained for PLP to mark centrioles and Hoechst stained to label DNA. Cell borders are traced with dashed lines. Bar, 5 µm. (B) PP2A inhibition by Mts, 29B, or Tws RNAi leads to centriole loss. After RNAi treatment, the number of PLP-immunostained centrioles per cell was measured. Graph shows the percentage of cells with the indicated number of centrioles per cell. Each number in a bar is the percent mean for two experiments (n = 500 cells/treatment). Asterisks mark significant differences (relative to control) for comparisons mentioned in the text (P < 0.02). Error bars indicate SD. (C) Mts cosediments with centrioles purified from mitotic S2 cells on a 20–70% sucrose gradient. Fractions (numbered) were immunoblotted for the indicated proteins. Asterisks mark the major centriole-containing fractions. (D) Tws protein is maximal during mitosis. (left) Graph of normalized endogenous Mts and Tws levels in asynchronous cells (Asynch) or after a 24-h drug-induced cell cycle arrest. Plotted values were determined from the anti-Mts and Tws immunoblots (right) shown. The graph and blots are representative examples of three independent experiments, all with similar results. α-Tubulin (α Tub) was used as a loading control. Wdb, Widerborst; Wrd, Well rounded.
Mentions: PP2A is a holoenzyme composed of three subunits: a catalytic C subunit (microtubule star [Mts] in Drosophila), a structural A subunit (Drosophila PP2A-29B), and a regulatory B subunit encoded by one of four fly genes (Tws/PR55, Widerborst, Well rounded, or PR72; Mayer-Jaekel et al., 1992; Uemura et al., 1993; Snaith et al., 1996; Hannus et al., 2002; Li et al., 2002; Viquez et al., 2006). To test whether PP2A is involved in centriole duplication, we used RNAi to deplete each protein individually (when antibodies were available, we confirmed depletion by immunoblotting; Fig. S3 A) and measured centriole numbers in S2 cells. Depletion of Mts or PP2A-29B resulted in loss of centrioles (Fig. 2 A), significantly increasing the percentage of cells with less than two centrioles compared with controls (Fig. 2 B). This effect was phenocopied by treating cells with the PP2A inhibitor okadaic acid (OA; Fig. 2, A and B). Depletion of only one regulatory subunit, Tws, also significantly decreased centriole number (Fig. 2 B). Prolonged Tws RNAi had no effect on cell cycle progression (Fig. S3 B) or mitotic index (control RNAi = 2.0%; Tws RNAi = 2.7%) but dramatically elevated the frequency of mitotic spindles lacking centrosomes (similar experiments with prolonged Mts or PP2A-29B RNAi led to significant cell death; Fig. S3 C). Consistent with a role in centriole duplication, Mts and PP2A-29B localize to mitotic centrioles in S2R+ cells (Dobbelaere et al., 2008). Likewise, we found a fraction of Mts copurified with centrioles isolated from mitotic S2 cells using sucrose gradient centrifugation (Fig. 2 C). Together, these results suggest that a PP2ATws complex is required for centriole duplication and is appropriately positioned on mitotic centrioles.

Bottom Line: However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown.However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification.We demonstrate that ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA.

ABSTRACT
Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. In this paper, we show that PP2A (Protein Phosphatase 2A(Twins)) counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. We demonstrate that ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification.

Show MeSH
Related in: MedlinePlus