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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

Drosophila Plk4 autophosphorylation promotes its own degradation. (A) Linear map of Drosophila Plk4 amino-terminus encoding the KD and the DRE. The red bar indicates the position of the conserved SBD (DSGXXT). (B) Lineup of the 50 amino acid DRE encoded by Plk4 family members. Serine and threonine residues are shown in red. Yellow box highlights the SBD. (C) Purified recombinant Plk4 kinase domain + DRE (wt-Plk4) but not kinase-dead (D156N point mutation of wt) Plk4 autophosphorylates in vitro. Coomassie-stained gel (top) and corresponding autoradiograph (bottom) are shown. (lanes 1 and 2) Plk4 phosphorylates itself and purified bovine myelin basic protein (MBP). (lane 3) Kinase-dead Plk4 lacks kinase activity. (lane 4) wt-Plk4 phosphorylates kinase-dead Plk4 in trans. (D) wt-Plk4 phosphorylates purified DRE fused to maltose-binding protein (MBP-DRE) in trans but does not phosphorylate purified maltose-binding protein. (E) A kinase-dead mutation in Plk4 inhibits its degradation in S2 cells. Anti-GFP immunoblot of S2 cell lysates shows that full-length kinase-dead Plk4-GFP is more stable than wt-Plk4-GFP, which is degraded and nearly undetectable. Inducible Plk4 constructs were cotransfected into S2 cells with Nlp-GFP (used as a loading control and driven by its endogenous promoter). Black/white lines indicate that intervening lanes have been spliced out.
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fig1: Drosophila Plk4 autophosphorylation promotes its own degradation. (A) Linear map of Drosophila Plk4 amino-terminus encoding the KD and the DRE. The red bar indicates the position of the conserved SBD (DSGXXT). (B) Lineup of the 50 amino acid DRE encoded by Plk4 family members. Serine and threonine residues are shown in red. Yellow box highlights the SBD. (C) Purified recombinant Plk4 kinase domain + DRE (wt-Plk4) but not kinase-dead (D156N point mutation of wt) Plk4 autophosphorylates in vitro. Coomassie-stained gel (top) and corresponding autoradiograph (bottom) are shown. (lanes 1 and 2) Plk4 phosphorylates itself and purified bovine myelin basic protein (MBP). (lane 3) Kinase-dead Plk4 lacks kinase activity. (lane 4) wt-Plk4 phosphorylates kinase-dead Plk4 in trans. (D) wt-Plk4 phosphorylates purified DRE fused to maltose-binding protein (MBP-DRE) in trans but does not phosphorylate purified maltose-binding protein. (E) A kinase-dead mutation in Plk4 inhibits its degradation in S2 cells. Anti-GFP immunoblot of S2 cell lysates shows that full-length kinase-dead Plk4-GFP is more stable than wt-Plk4-GFP, which is degraded and nearly undetectable. Inducible Plk4 constructs were cotransfected into S2 cells with Nlp-GFP (used as a loading control and driven by its endogenous promoter). Black/white lines indicate that intervening lanes have been spliced out.

Mentions: Previously, we found that the Plk4 protein accumulates during mitosis in cultured Drosophila S2 cells (Fig. S1; Rogers et al., 2009). At this time, Plk4 associates with centrioles and endows them with the competence to duplicate. During interphase, Plk4 is recognized by SCFSlimb ubiquitin ligase, and its consequent degradation is crucial in blocking centriole reduplication and preventing mother centrioles from assembling multiple daughters (Cunha-Ferreira et al., 2009; Rogers et al., 2009). Phosphorylation of the conserved Slimb-binding domain (SBD) within Plk4 promotes recognition by Slimb, and therefore, we set out to identify the kinases that prime Plk4 for degradation. Recently, it was reported that mammalian Plk4 autophosphorylates a region downstream of its kinase domain (KD; ∼50 amino acids containing the SBD) to promote its destruction by SCFβTrCP/Slimb (Guderian et al., 2010; Holland et al., 2010). Drosophila Plk4 also contains a serine-rich region surrounding its SBD (denoted here as the downstream regulatory element [DRE]; Fig. 1, A and B). To test whether fly Plk4 utilizes a similar autophosphorylation mechanism, we generated recombinant Plk4 containing the KD and DRE (Plk4-KD). We found that Plk4-KD can autophosphorylate in vitro and can phosphorylate kinase-dead Plk4-KD in trans (Fig. 1 C). As with mammalian Plk4 (Leung et al., 2007; Holland et al., 2010; Sillibourne et al., 2010), the DRE of Drosophila Plk4 is a target for autophosphorylation, as Plk4-KD phosphorylates purified DRE (Fig. 1 D). Furthermore, we found that the Plk4 kinase activity is needed for its degradation because kinase-dead Plk4 expressed in S2 cells is more stable than wild-type (wt) Plk4 (Fig. 1 E). These results support the hypothesis that Plk4 autophosphorylation promotes its destruction but do not exclude the possibility that other kinases also regulate Plk4 levels. Therefore, we performed an RNAi-based screen of the Drosophila kinome using S2 cells transfected with inducible Plk4-GFP and measured Plk4 levels by quantitative anti-GFP immunoblots (Table S2). As with Slimb RNAi treatment, depletion of the kinase responsible for Slimb recognition should cause a dramatic elevation in Plk4 levels. However, no such kinase was identified in our screen (Fig. S2). Collectively, these results demonstrate that Plk4 autophosphorylation of the DRE is an evolutionarily conserved mechanism that regulates Plk4 stability and that it is unlikely that other kinases influence Slimb binding.


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)

Drosophila Plk4 autophosphorylation promotes its own degradation. (A) Linear map of Drosophila Plk4 amino-terminus encoding the KD and the DRE. The red bar indicates the position of the conserved SBD (DSGXXT). (B) Lineup of the 50 amino acid DRE encoded by Plk4 family members. Serine and threonine residues are shown in red. Yellow box highlights the SBD. (C) Purified recombinant Plk4 kinase domain + DRE (wt-Plk4) but not kinase-dead (D156N point mutation of wt) Plk4 autophosphorylates in vitro. Coomassie-stained gel (top) and corresponding autoradiograph (bottom) are shown. (lanes 1 and 2) Plk4 phosphorylates itself and purified bovine myelin basic protein (MBP). (lane 3) Kinase-dead Plk4 lacks kinase activity. (lane 4) wt-Plk4 phosphorylates kinase-dead Plk4 in trans. (D) wt-Plk4 phosphorylates purified DRE fused to maltose-binding protein (MBP-DRE) in trans but does not phosphorylate purified maltose-binding protein. (E) A kinase-dead mutation in Plk4 inhibits its degradation in S2 cells. Anti-GFP immunoblot of S2 cell lysates shows that full-length kinase-dead Plk4-GFP is more stable than wt-Plk4-GFP, which is degraded and nearly undetectable. Inducible Plk4 constructs were cotransfected into S2 cells with Nlp-GFP (used as a loading control and driven by its endogenous promoter). Black/white lines indicate that intervening lanes have been spliced out.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Drosophila Plk4 autophosphorylation promotes its own degradation. (A) Linear map of Drosophila Plk4 amino-terminus encoding the KD and the DRE. The red bar indicates the position of the conserved SBD (DSGXXT). (B) Lineup of the 50 amino acid DRE encoded by Plk4 family members. Serine and threonine residues are shown in red. Yellow box highlights the SBD. (C) Purified recombinant Plk4 kinase domain + DRE (wt-Plk4) but not kinase-dead (D156N point mutation of wt) Plk4 autophosphorylates in vitro. Coomassie-stained gel (top) and corresponding autoradiograph (bottom) are shown. (lanes 1 and 2) Plk4 phosphorylates itself and purified bovine myelin basic protein (MBP). (lane 3) Kinase-dead Plk4 lacks kinase activity. (lane 4) wt-Plk4 phosphorylates kinase-dead Plk4 in trans. (D) wt-Plk4 phosphorylates purified DRE fused to maltose-binding protein (MBP-DRE) in trans but does not phosphorylate purified maltose-binding protein. (E) A kinase-dead mutation in Plk4 inhibits its degradation in S2 cells. Anti-GFP immunoblot of S2 cell lysates shows that full-length kinase-dead Plk4-GFP is more stable than wt-Plk4-GFP, which is degraded and nearly undetectable. Inducible Plk4 constructs were cotransfected into S2 cells with Nlp-GFP (used as a loading control and driven by its endogenous promoter). Black/white lines indicate that intervening lanes have been spliced out.
Mentions: Previously, we found that the Plk4 protein accumulates during mitosis in cultured Drosophila S2 cells (Fig. S1; Rogers et al., 2009). At this time, Plk4 associates with centrioles and endows them with the competence to duplicate. During interphase, Plk4 is recognized by SCFSlimb ubiquitin ligase, and its consequent degradation is crucial in blocking centriole reduplication and preventing mother centrioles from assembling multiple daughters (Cunha-Ferreira et al., 2009; Rogers et al., 2009). Phosphorylation of the conserved Slimb-binding domain (SBD) within Plk4 promotes recognition by Slimb, and therefore, we set out to identify the kinases that prime Plk4 for degradation. Recently, it was reported that mammalian Plk4 autophosphorylates a region downstream of its kinase domain (KD; ∼50 amino acids containing the SBD) to promote its destruction by SCFβTrCP/Slimb (Guderian et al., 2010; Holland et al., 2010). Drosophila Plk4 also contains a serine-rich region surrounding its SBD (denoted here as the downstream regulatory element [DRE]; Fig. 1, A and B). To test whether fly Plk4 utilizes a similar autophosphorylation mechanism, we generated recombinant Plk4 containing the KD and DRE (Plk4-KD). We found that Plk4-KD can autophosphorylate in vitro and can phosphorylate kinase-dead Plk4-KD in trans (Fig. 1 C). As with mammalian Plk4 (Leung et al., 2007; Holland et al., 2010; Sillibourne et al., 2010), the DRE of Drosophila Plk4 is a target for autophosphorylation, as Plk4-KD phosphorylates purified DRE (Fig. 1 D). Furthermore, we found that the Plk4 kinase activity is needed for its degradation because kinase-dead Plk4 expressed in S2 cells is more stable than wild-type (wt) Plk4 (Fig. 1 E). These results support the hypothesis that Plk4 autophosphorylation promotes its destruction but do not exclude the possibility that other kinases also regulate Plk4 levels. Therefore, we performed an RNAi-based screen of the Drosophila kinome using S2 cells transfected with inducible Plk4-GFP and measured Plk4 levels by quantitative anti-GFP immunoblots (Table S2). As with Slimb RNAi treatment, depletion of the kinase responsible for Slimb recognition should cause a dramatic elevation in Plk4 levels. However, no such kinase was identified in our screen (Fig. S2). Collectively, these results demonstrate that Plk4 autophosphorylation of the DRE is an evolutionarily conserved mechanism that regulates Plk4 stability and that it is unlikely that other kinases influence Slimb binding.

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