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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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Related in: MedlinePlus

Ectopic Tws expression is sufficient to stabilize Plk4 and promote centriole amplification. (A) Immunoblots of S2 cell lysates showing that Tws-GFP overexpression stabilizes Plk4-GFP without affecting Slimb levels. S2 cells were cotransfected with inducible Plk4-GFP and inducible GFP (first lane) or Tws-GFP (second lane) expression constructs. After a 24-h recovery period, gene expression was induced with 1 mM CuSO4 for 20 h. Cell lysates were then prepared and immunoblotted for GFP, endogenous Slimb, and α-tubulin (α Tub; loading control). (B) The extent of Plk4-GFP stabilization is dose dependent on Tws-GFP. S2 cells were cotransfected with Plk4-GFP (driven by the weak, constitutive Drosophila SAS-6 promoter) and either GFP (negative control) or Tws-GFP, each controlled by the copper-inducible metallothionein promoter. Cells were incubated with 0, 0.5, 1, or 2 mM CuSO4 for 24 h, and cell lysates were probed by GFP immunoblotting. (C) Immunoblot of S2 cell lysates showing that Plk4-GFP is also stabilized by human Tws (HsTws; PR55-α) overexpression. (D) Tws-mCherry expression drives abnormal accumulation of Plk4-GFP on centrioles (anti-D-PLP) in interphase S2 cells. Insets show centrioles (dashed boxes) at higher magnification. Bars, 5 µm. (E) Tws-GFP overexpression promotes centriole amplification. Graph shows the percentage of transgene-expressing cells containing the indicated number of centrioles; means (numbers) derived from three experiments (n = 600 cells/treatment). *, P < 0.003 (treated conditions compared with GFP control). (F) Tws-GFP overexpression increases the frequency of multipolar spindles. S2 cells expressing GFP or Tws-GFP were immunostained for centrioles (anti-PLP, red) and α-tubulin (green). DNA (blue) is Hoechst stained. Bar, 2.5 µm. Graph shows mean percentages (numbers) of mitotic cells with multipolar spindles (two experiments; n = 76 cells/treatment). *, P < 0.01. Error bars indicate SD.
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fig4: Ectopic Tws expression is sufficient to stabilize Plk4 and promote centriole amplification. (A) Immunoblots of S2 cell lysates showing that Tws-GFP overexpression stabilizes Plk4-GFP without affecting Slimb levels. S2 cells were cotransfected with inducible Plk4-GFP and inducible GFP (first lane) or Tws-GFP (second lane) expression constructs. After a 24-h recovery period, gene expression was induced with 1 mM CuSO4 for 20 h. Cell lysates were then prepared and immunoblotted for GFP, endogenous Slimb, and α-tubulin (α Tub; loading control). (B) The extent of Plk4-GFP stabilization is dose dependent on Tws-GFP. S2 cells were cotransfected with Plk4-GFP (driven by the weak, constitutive Drosophila SAS-6 promoter) and either GFP (negative control) or Tws-GFP, each controlled by the copper-inducible metallothionein promoter. Cells were incubated with 0, 0.5, 1, or 2 mM CuSO4 for 24 h, and cell lysates were probed by GFP immunoblotting. (C) Immunoblot of S2 cell lysates showing that Plk4-GFP is also stabilized by human Tws (HsTws; PR55-α) overexpression. (D) Tws-mCherry expression drives abnormal accumulation of Plk4-GFP on centrioles (anti-D-PLP) in interphase S2 cells. Insets show centrioles (dashed boxes) at higher magnification. Bars, 5 µm. (E) Tws-GFP overexpression promotes centriole amplification. Graph shows the percentage of transgene-expressing cells containing the indicated number of centrioles; means (numbers) derived from three experiments (n = 600 cells/treatment). *, P < 0.003 (treated conditions compared with GFP control). (F) Tws-GFP overexpression increases the frequency of multipolar spindles. S2 cells expressing GFP or Tws-GFP were immunostained for centrioles (anti-PLP, red) and α-tubulin (green). DNA (blue) is Hoechst stained. Bar, 2.5 µm. Graph shows mean percentages (numbers) of mitotic cells with multipolar spindles (two experiments; n = 76 cells/treatment). *, P < 0.01. Error bars indicate SD.

Mentions: Because Tws protein levels peak during mitosis when Plk4 levels are highest (Fig. 2 D), we tested whether overexpression of this regulatory subunit alone is sufficient to stabilize Plk4 during periods when Plk4 is normally degraded. Tws-GFP (or GFP) and Plk4-GFP were coexpressed in S2 cells, and their levels were monitored with GFP immunoblots. Although control GFP overexpression had no effect on Plk4-GFP levels, Tws-GFP overexpression dramatically increased Plk4-GFP levels in asynchronous cells in a dose-dependent manner (Fig. 4, A and B). The effect was not caused by mitotic arrest (mitotic index: GFP, 2.7%; Tws-GFP, 3.2%) nor caused by a decrease in Slimb levels, which remained unchanged (Fig. 4 A). A similar result was observed in S2 cells overexpressing human Tws (which shares 79% amino acid identity with fly Tws), in which Plk4-GFP levels increased by fourfold (Fig. 4 C), suggesting PP2ATws may function analogously in human cells to stabilize Plk4.


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)

Ectopic Tws expression is sufficient to stabilize Plk4 and promote centriole amplification. (A) Immunoblots of S2 cell lysates showing that Tws-GFP overexpression stabilizes Plk4-GFP without affecting Slimb levels. S2 cells were cotransfected with inducible Plk4-GFP and inducible GFP (first lane) or Tws-GFP (second lane) expression constructs. After a 24-h recovery period, gene expression was induced with 1 mM CuSO4 for 20 h. Cell lysates were then prepared and immunoblotted for GFP, endogenous Slimb, and α-tubulin (α Tub; loading control). (B) The extent of Plk4-GFP stabilization is dose dependent on Tws-GFP. S2 cells were cotransfected with Plk4-GFP (driven by the weak, constitutive Drosophila SAS-6 promoter) and either GFP (negative control) or Tws-GFP, each controlled by the copper-inducible metallothionein promoter. Cells were incubated with 0, 0.5, 1, or 2 mM CuSO4 for 24 h, and cell lysates were probed by GFP immunoblotting. (C) Immunoblot of S2 cell lysates showing that Plk4-GFP is also stabilized by human Tws (HsTws; PR55-α) overexpression. (D) Tws-mCherry expression drives abnormal accumulation of Plk4-GFP on centrioles (anti-D-PLP) in interphase S2 cells. Insets show centrioles (dashed boxes) at higher magnification. Bars, 5 µm. (E) Tws-GFP overexpression promotes centriole amplification. Graph shows the percentage of transgene-expressing cells containing the indicated number of centrioles; means (numbers) derived from three experiments (n = 600 cells/treatment). *, P < 0.003 (treated conditions compared with GFP control). (F) Tws-GFP overexpression increases the frequency of multipolar spindles. S2 cells expressing GFP or Tws-GFP were immunostained for centrioles (anti-PLP, red) and α-tubulin (green). DNA (blue) is Hoechst stained. Bar, 2.5 µm. Graph shows mean percentages (numbers) of mitotic cells with multipolar spindles (two experiments; n = 76 cells/treatment). *, P < 0.01. Error bars indicate SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig4: Ectopic Tws expression is sufficient to stabilize Plk4 and promote centriole amplification. (A) Immunoblots of S2 cell lysates showing that Tws-GFP overexpression stabilizes Plk4-GFP without affecting Slimb levels. S2 cells were cotransfected with inducible Plk4-GFP and inducible GFP (first lane) or Tws-GFP (second lane) expression constructs. After a 24-h recovery period, gene expression was induced with 1 mM CuSO4 for 20 h. Cell lysates were then prepared and immunoblotted for GFP, endogenous Slimb, and α-tubulin (α Tub; loading control). (B) The extent of Plk4-GFP stabilization is dose dependent on Tws-GFP. S2 cells were cotransfected with Plk4-GFP (driven by the weak, constitutive Drosophila SAS-6 promoter) and either GFP (negative control) or Tws-GFP, each controlled by the copper-inducible metallothionein promoter. Cells were incubated with 0, 0.5, 1, or 2 mM CuSO4 for 24 h, and cell lysates were probed by GFP immunoblotting. (C) Immunoblot of S2 cell lysates showing that Plk4-GFP is also stabilized by human Tws (HsTws; PR55-α) overexpression. (D) Tws-mCherry expression drives abnormal accumulation of Plk4-GFP on centrioles (anti-D-PLP) in interphase S2 cells. Insets show centrioles (dashed boxes) at higher magnification. Bars, 5 µm. (E) Tws-GFP overexpression promotes centriole amplification. Graph shows the percentage of transgene-expressing cells containing the indicated number of centrioles; means (numbers) derived from three experiments (n = 600 cells/treatment). *, P < 0.003 (treated conditions compared with GFP control). (F) Tws-GFP overexpression increases the frequency of multipolar spindles. S2 cells expressing GFP or Tws-GFP were immunostained for centrioles (anti-PLP, red) and α-tubulin (green). DNA (blue) is Hoechst stained. Bar, 2.5 µm. Graph shows mean percentages (numbers) of mitotic cells with multipolar spindles (two experiments; n = 76 cells/treatment). *, P < 0.01. Error bars indicate SD.
Mentions: Because Tws protein levels peak during mitosis when Plk4 levels are highest (Fig. 2 D), we tested whether overexpression of this regulatory subunit alone is sufficient to stabilize Plk4 during periods when Plk4 is normally degraded. Tws-GFP (or GFP) and Plk4-GFP were coexpressed in S2 cells, and their levels were monitored with GFP immunoblots. Although control GFP overexpression had no effect on Plk4-GFP levels, Tws-GFP overexpression dramatically increased Plk4-GFP levels in asynchronous cells in a dose-dependent manner (Fig. 4, A and B). The effect was not caused by mitotic arrest (mitotic index: GFP, 2.7%; Tws-GFP, 3.2%) nor caused by a decrease in Slimb levels, which remained unchanged (Fig. 4 A). A similar result was observed in S2 cells overexpressing human Tws (which shares 79% amino acid identity with fly Tws), in which Plk4-GFP levels increased by fourfold (Fig. 4 C), suggesting PP2ATws may function analogously in human cells to stabilize Plk4.

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