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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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Tumor-promoting SV40 ST mimics Tws function to stabilize Plk4 and amplify centrioles. (A) S2 cells overexpressing GFP or ST-GFP (green) immunostained for PLP (red) to mark centrioles. DNA (blue) is Hoechst stained. (B) ST-GFP overexpression increases centriole numbers but not in the presence of OA. ST-GFP expression also rescues centriole loss caused by depletion of Tws but not Plk4. Centrioles were counted after 6-d RNAi or 24-h OA treatment in cells transfected with inducible GFP or ST-GFP (expression was induced during the last 2 d). Mean values (numbers) of two experiments are shown (n = 600 cells/treatment). Asterisks indicate significant differences (P < 0.05) between compared treatments mentioned in the text. Error bars indicate SD. Ctnrl, control. (C and D) Immunoblots show that ST-GFP overexpression stabilizes Plk4-GFP in S2 cells to levels similar to Plk4-SBM-GFP, without affecting endogenous Slimb levels (C) and in a dose-dependent manner (D; treatment protocol similar to that described in Fig. 4 B). α Tub, α-tubulin. (E) ST-V5 expression drives accumulation of Plk4-GFP on centrioles (anti-PLP) in interphase S2 cells. Insets show centrioles (dashed boxes) at higher magnification. Bars, 5 µm.
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fig5: Tumor-promoting SV40 ST mimics Tws function to stabilize Plk4 and amplify centrioles. (A) S2 cells overexpressing GFP or ST-GFP (green) immunostained for PLP (red) to mark centrioles. DNA (blue) is Hoechst stained. (B) ST-GFP overexpression increases centriole numbers but not in the presence of OA. ST-GFP expression also rescues centriole loss caused by depletion of Tws but not Plk4. Centrioles were counted after 6-d RNAi or 24-h OA treatment in cells transfected with inducible GFP or ST-GFP (expression was induced during the last 2 d). Mean values (numbers) of two experiments are shown (n = 600 cells/treatment). Asterisks indicate significant differences (P < 0.05) between compared treatments mentioned in the text. Error bars indicate SD. Ctnrl, control. (C and D) Immunoblots show that ST-GFP overexpression stabilizes Plk4-GFP in S2 cells to levels similar to Plk4-SBM-GFP, without affecting endogenous Slimb levels (C) and in a dose-dependent manner (D; treatment protocol similar to that described in Fig. 4 B). α Tub, α-tubulin. (E) ST-V5 expression drives accumulation of Plk4-GFP on centrioles (anti-PLP) in interphase S2 cells. Insets show centrioles (dashed boxes) at higher magnification. Bars, 5 µm.

Mentions: Based on these findings, we propose that centriole duplication is regulated by a dynamic mechanism governing Plk4 stability: Plk4 autophosphorylation of its DRE promotes SCFSlimb-mediated degradation during interphase to block centriole amplification, but the phosphorylated state is reversed by mitotic PP2ATws to stabilize Plk4 and enable centriole duplication. As a proof of principle study, we examined whether perturbation of this mechanism could account for centrosome amplification observed during oncogenic transformation by using the ST of the tumor-promoting virus SV40 (Kotadia et al., 2008). ST can act as a potent PP2A inhibitor (Arroyo and Hahn, 2005). By directly binding to the PP2A structural A subunit, ST competes with and displaces endogenous regulatory subunits, thereby preventing the dephosphorylation of PP2A substrates (Chen et al., 2007b; Cho et al., 2007). Not surprisingly, given their high degree of conservation, ST also binds the Drosophila structural subunit PP2A-29B (Kotadia et al., 2008). However, regarding ST’s effect on centriole duplication, the notion that ST inhibits PP2A is paradoxical because ST expression promotes centrosome amplification in human U2OS cells, cultured fly Kc cells, and fly embryos (Kotadia et al., 2008), whereas PP2A depletion actually eliminates centrosomes in fly cells (Chen et al., 2007a). To resolve this issue, we tested the hypothesis that ST does not inhibit all PP2A functions but can instead act as a surrogate regulatory subunit to mimic the activity of Tws by stabilizing Plk4 and, thus, drive centriole amplification. We found that ST-GFP overexpression in S2 cells increased the percentage of cells containing greater than two centrioles (Fig. 5, A and B). Notably, the effect is PP2A dependent because OA treatment of ST-GFP–expressing cells blocks centriole amplification (Fig. 5 B). Therefore, the centriole amplification that follows ST expression requires PP2A activity and suggests that ST does not inhibit all PP2A functions. This result is surprising given ST’s reported inhibitory effects on PP2A activity.


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)

Tumor-promoting SV40 ST mimics Tws function to stabilize Plk4 and amplify centrioles. (A) S2 cells overexpressing GFP or ST-GFP (green) immunostained for PLP (red) to mark centrioles. DNA (blue) is Hoechst stained. (B) ST-GFP overexpression increases centriole numbers but not in the presence of OA. ST-GFP expression also rescues centriole loss caused by depletion of Tws but not Plk4. Centrioles were counted after 6-d RNAi or 24-h OA treatment in cells transfected with inducible GFP or ST-GFP (expression was induced during the last 2 d). Mean values (numbers) of two experiments are shown (n = 600 cells/treatment). Asterisks indicate significant differences (P < 0.05) between compared treatments mentioned in the text. Error bars indicate SD. Ctnrl, control. (C and D) Immunoblots show that ST-GFP overexpression stabilizes Plk4-GFP in S2 cells to levels similar to Plk4-SBM-GFP, without affecting endogenous Slimb levels (C) and in a dose-dependent manner (D; treatment protocol similar to that described in Fig. 4 B). α Tub, α-tubulin. (E) ST-V5 expression drives accumulation of Plk4-GFP on centrioles (anti-PLP) in interphase S2 cells. Insets show centrioles (dashed boxes) at higher magnification. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Tumor-promoting SV40 ST mimics Tws function to stabilize Plk4 and amplify centrioles. (A) S2 cells overexpressing GFP or ST-GFP (green) immunostained for PLP (red) to mark centrioles. DNA (blue) is Hoechst stained. (B) ST-GFP overexpression increases centriole numbers but not in the presence of OA. ST-GFP expression also rescues centriole loss caused by depletion of Tws but not Plk4. Centrioles were counted after 6-d RNAi or 24-h OA treatment in cells transfected with inducible GFP or ST-GFP (expression was induced during the last 2 d). Mean values (numbers) of two experiments are shown (n = 600 cells/treatment). Asterisks indicate significant differences (P < 0.05) between compared treatments mentioned in the text. Error bars indicate SD. Ctnrl, control. (C and D) Immunoblots show that ST-GFP overexpression stabilizes Plk4-GFP in S2 cells to levels similar to Plk4-SBM-GFP, without affecting endogenous Slimb levels (C) and in a dose-dependent manner (D; treatment protocol similar to that described in Fig. 4 B). α Tub, α-tubulin. (E) ST-V5 expression drives accumulation of Plk4-GFP on centrioles (anti-PLP) in interphase S2 cells. Insets show centrioles (dashed boxes) at higher magnification. Bars, 5 µm.
Mentions: Based on these findings, we propose that centriole duplication is regulated by a dynamic mechanism governing Plk4 stability: Plk4 autophosphorylation of its DRE promotes SCFSlimb-mediated degradation during interphase to block centriole amplification, but the phosphorylated state is reversed by mitotic PP2ATws to stabilize Plk4 and enable centriole duplication. As a proof of principle study, we examined whether perturbation of this mechanism could account for centrosome amplification observed during oncogenic transformation by using the ST of the tumor-promoting virus SV40 (Kotadia et al., 2008). ST can act as a potent PP2A inhibitor (Arroyo and Hahn, 2005). By directly binding to the PP2A structural A subunit, ST competes with and displaces endogenous regulatory subunits, thereby preventing the dephosphorylation of PP2A substrates (Chen et al., 2007b; Cho et al., 2007). Not surprisingly, given their high degree of conservation, ST also binds the Drosophila structural subunit PP2A-29B (Kotadia et al., 2008). However, regarding ST’s effect on centriole duplication, the notion that ST inhibits PP2A is paradoxical because ST expression promotes centrosome amplification in human U2OS cells, cultured fly Kc cells, and fly embryos (Kotadia et al., 2008), whereas PP2A depletion actually eliminates centrosomes in fly cells (Chen et al., 2007a). To resolve this issue, we tested the hypothesis that ST does not inhibit all PP2A functions but can instead act as a surrogate regulatory subunit to mimic the activity of Tws by stabilizing Plk4 and, thus, drive centriole amplification. We found that ST-GFP overexpression in S2 cells increased the percentage of cells containing greater than two centrioles (Fig. 5, A and B). Notably, the effect is PP2A dependent because OA treatment of ST-GFP–expressing cells blocks centriole amplification (Fig. 5 B). Therefore, the centriole amplification that follows ST expression requires PP2A activity and suggests that ST does not inhibit all PP2A functions. This result is surprising given ST’s reported inhibitory effects on PP2A activity.

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