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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 stabilizes Plk4 to promote centriole duplication. (A) Immunoblots of 6-d RNAi-treated S2 cells demonstrating knockdown of target proteins. α Tub, α-tubulin; Cntrl, control. (B) Loss of centrioles by Tws RNAi is rescued by codepletion of Slimb. Each mean percentage of cells (numbers) is derived from two experiments (n = 598 cells/treatment). Asterisk indicates significant difference (P < 0.02) between compared treatments mentioned in the text. (C) Low expression of nondegradable Plk4-SBM-GFP also rescues the centriole loss by Tws-RNAi. Each mean percentage (numbers) is derived from three experiments (n = 900 cells/treatment). *, P < 0.04. (D) PP2A is required to stabilize Plk4. S2 cells overexpressing Plk4-GFP were treated with colchicine or OA for 24 h as indicated, and lysates were probed for GFP. (E) PP2A dephosphorylates Plk4-SBM-GFP in cells. S2 cells expressing Plk4-SBM-GFP were treated with OA for 24 h, and their lysates were immunoblotted for GFP. Note the clear shift in mobility of Plk4-SBM after OA treatment (arrowheads), consistent with Plk4-SBM being hyperphosphorylated after PP2A inhibition. (F) PP2A dephosphorylates Plk4 in vitro. Human PP2A dephosphorylates autophosphorylated Plk4-KD + DRE-(His)6 protein (Plk4-KD) but is inhibited by OA. (top) Autoradiogram; (bottom) Plk4 immunoblot. Black lines indicate that intervening lanes have been spliced out. Error bars indicate SD.
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fig3: PP2ATws stabilizes Plk4 to promote centriole duplication. (A) Immunoblots of 6-d RNAi-treated S2 cells demonstrating knockdown of target proteins. α Tub, α-tubulin; Cntrl, control. (B) Loss of centrioles by Tws RNAi is rescued by codepletion of Slimb. Each mean percentage of cells (numbers) is derived from two experiments (n = 598 cells/treatment). Asterisk indicates significant difference (P < 0.02) between compared treatments mentioned in the text. (C) Low expression of nondegradable Plk4-SBM-GFP also rescues the centriole loss by Tws-RNAi. Each mean percentage (numbers) is derived from three experiments (n = 900 cells/treatment). *, P < 0.04. (D) PP2A is required to stabilize Plk4. S2 cells overexpressing Plk4-GFP were treated with colchicine or OA for 24 h as indicated, and lysates were probed for GFP. (E) PP2A dephosphorylates Plk4-SBM-GFP in cells. S2 cells expressing Plk4-SBM-GFP were treated with OA for 24 h, and their lysates were immunoblotted for GFP. Note the clear shift in mobility of Plk4-SBM after OA treatment (arrowheads), consistent with Plk4-SBM being hyperphosphorylated after PP2A inhibition. (F) PP2A dephosphorylates Plk4 in vitro. Human PP2A dephosphorylates autophosphorylated Plk4-KD + DRE-(His)6 protein (Plk4-KD) but is inhibited by OA. (top) Autoradiogram; (bottom) Plk4 immunoblot. Black lines indicate that intervening lanes have been spliced out. Error bars indicate SD.

Mentions: We next tested whether Tws is involved in the pathway regulating Plk4 stability. Depletion of Slimb increases both Plk4 levels and the percentage of cells with greater than two centrioles (Cunha-Ferreira et al., 2009; Rogers et al., 2009). Therefore, if loss of centrioles after PP2ATws depletion is caused by Plk4 degradation, this effect should be rescued by codepletion of Slimb because Slimb mediates Plk4 degradation. Indeed, we found that whereas Tws RNAi reduced centriole number, Slimb/Tws co-RNAi (which effectively depletes both proteins; Fig. 3 A) completely reversed the effect and significantly decreased the percentage of cells containing less than two centrioles while increasing cells with greater than two centrioles (Fig. 3 B). Therefore, instead of being required for downstream steps in centriole assembly, PP2ATws likely acts upstream of Plk4 and Slimb.


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 stabilizes Plk4 to promote centriole duplication. (A) Immunoblots of 6-d RNAi-treated S2 cells demonstrating knockdown of target proteins. α Tub, α-tubulin; Cntrl, control. (B) Loss of centrioles by Tws RNAi is rescued by codepletion of Slimb. Each mean percentage of cells (numbers) is derived from two experiments (n = 598 cells/treatment). Asterisk indicates significant difference (P < 0.02) between compared treatments mentioned in the text. (C) Low expression of nondegradable Plk4-SBM-GFP also rescues the centriole loss by Tws-RNAi. Each mean percentage (numbers) is derived from three experiments (n = 900 cells/treatment). *, P < 0.04. (D) PP2A is required to stabilize Plk4. S2 cells overexpressing Plk4-GFP were treated with colchicine or OA for 24 h as indicated, and lysates were probed for GFP. (E) PP2A dephosphorylates Plk4-SBM-GFP in cells. S2 cells expressing Plk4-SBM-GFP were treated with OA for 24 h, and their lysates were immunoblotted for GFP. Note the clear shift in mobility of Plk4-SBM after OA treatment (arrowheads), consistent with Plk4-SBM being hyperphosphorylated after PP2A inhibition. (F) PP2A dephosphorylates Plk4 in vitro. Human PP2A dephosphorylates autophosphorylated Plk4-KD + DRE-(His)6 protein (Plk4-KD) but is inhibited by OA. (top) Autoradiogram; (bottom) Plk4 immunoblot. Black lines indicate that intervening lanes have been spliced out. Error bars indicate SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3198173&req=5

fig3: PP2ATws stabilizes Plk4 to promote centriole duplication. (A) Immunoblots of 6-d RNAi-treated S2 cells demonstrating knockdown of target proteins. α Tub, α-tubulin; Cntrl, control. (B) Loss of centrioles by Tws RNAi is rescued by codepletion of Slimb. Each mean percentage of cells (numbers) is derived from two experiments (n = 598 cells/treatment). Asterisk indicates significant difference (P < 0.02) between compared treatments mentioned in the text. (C) Low expression of nondegradable Plk4-SBM-GFP also rescues the centriole loss by Tws-RNAi. Each mean percentage (numbers) is derived from three experiments (n = 900 cells/treatment). *, P < 0.04. (D) PP2A is required to stabilize Plk4. S2 cells overexpressing Plk4-GFP were treated with colchicine or OA for 24 h as indicated, and lysates were probed for GFP. (E) PP2A dephosphorylates Plk4-SBM-GFP in cells. S2 cells expressing Plk4-SBM-GFP were treated with OA for 24 h, and their lysates were immunoblotted for GFP. Note the clear shift in mobility of Plk4-SBM after OA treatment (arrowheads), consistent with Plk4-SBM being hyperphosphorylated after PP2A inhibition. (F) PP2A dephosphorylates Plk4 in vitro. Human PP2A dephosphorylates autophosphorylated Plk4-KD + DRE-(His)6 protein (Plk4-KD) but is inhibited by OA. (top) Autoradiogram; (bottom) Plk4 immunoblot. Black lines indicate that intervening lanes have been spliced out. Error bars indicate SD.
Mentions: We next tested whether Tws is involved in the pathway regulating Plk4 stability. Depletion of Slimb increases both Plk4 levels and the percentage of cells with greater than two centrioles (Cunha-Ferreira et al., 2009; Rogers et al., 2009). Therefore, if loss of centrioles after PP2ATws depletion is caused by Plk4 degradation, this effect should be rescued by codepletion of Slimb because Slimb mediates Plk4 degradation. Indeed, we found that whereas Tws RNAi reduced centriole number, Slimb/Tws co-RNAi (which effectively depletes both proteins; Fig. 3 A) completely reversed the effect and significantly decreased the percentage of cells containing less than two centrioles while increasing cells with greater than two centrioles (Fig. 3 B). Therefore, instead of being required for downstream steps in centriole assembly, PP2ATws likely acts upstream of Plk4 and Slimb.

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