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The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication.

Rogers GC, Rusan NM, Roberts DM, Peifer M, Rogers SL - J. Cell Biol. (2009)

Bottom Line: We found that Plk4 binds to Slimb and is an SCF(Slimb) target.Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase.Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
Restricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCF(Slimb) as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCF(Slimb) target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

Show MeSH
Plk4 is degraded in a Slimb-dependent manner and is stabilized by perturbing its interaction with Slimb. (A) Plk4 family showing the conserved kinase domain (gray), Polo box motif (striped boxes), and Slimb-binding consensus (black bars). The S293A/T297A SBM should be nondegradable. (B) Preimmune control and anti-Slimb immunoprecipitates from stable S2 cell lysates expressing Plk4-myc were probed for anti-Slimb, SkpA, and myc. (C) Control and anti-GFP immunoprecipitates from S2 cell lysates transiently expressing either inducible wild-type Plk4-EGFP or Plk4-SBM–EGFP were probed for endogenous Slimb. IP, immunoprecipitation. (D) Anti-GFP immunoprecipitates from S2 cell lysates transiently expressing triple Flag-ubiquitin and either inducible wild-type Plk4-EGFP or Plk4-SBM–EGFP were probed with anti-GFP (left) and anti-Flag (right) antibody. IB, immunoblot. (E) Anti-GFP immunoblots of lysates prepared from stable SAS-6p–driven Plk4-EGFP that were RNAi treated for the indicated protein for 7 d. (F) Plk4 is phosphorylated. (left) Anti-GFP immunoblots of lysates from control or Slimb-depleted cells transiently expressing inducible Plk4-EGFP. Plk4 accumulates as a doublet (arrowheads) after slimb RNAi. (right) Anti-GFP immunoprecipitates from day 4 Slimb-depleted cells transiently expressing inducible Plk4-EGFP were mock or alkaline phosphatase treated. Plk4 shifts from a broad band (bracket) to a faster migrating single polypeptide (arrowhead). (D–F) Molecular mass is indicated in kilodaltons. (G) Anti-GFP immunoblots showing the levels of transiently expressed SAS-6p–driven Plk4-EGFP and Plk4-SBM–EGFP in 24-h drug-induced cell cycle–arrested cells. Cotransfected Nlp-EGFP was used as a loading control. (H) Anti-GFP immunoblots showing the levels of 4 h–induced Plk4-EGFP and Plk4-SBM–EGFP expression in drug-induced cell cycle–arrested cells. (E and H) α-Tubulin was used as a loading control.
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fig3: Plk4 is degraded in a Slimb-dependent manner and is stabilized by perturbing its interaction with Slimb. (A) Plk4 family showing the conserved kinase domain (gray), Polo box motif (striped boxes), and Slimb-binding consensus (black bars). The S293A/T297A SBM should be nondegradable. (B) Preimmune control and anti-Slimb immunoprecipitates from stable S2 cell lysates expressing Plk4-myc were probed for anti-Slimb, SkpA, and myc. (C) Control and anti-GFP immunoprecipitates from S2 cell lysates transiently expressing either inducible wild-type Plk4-EGFP or Plk4-SBM–EGFP were probed for endogenous Slimb. IP, immunoprecipitation. (D) Anti-GFP immunoprecipitates from S2 cell lysates transiently expressing triple Flag-ubiquitin and either inducible wild-type Plk4-EGFP or Plk4-SBM–EGFP were probed with anti-GFP (left) and anti-Flag (right) antibody. IB, immunoblot. (E) Anti-GFP immunoblots of lysates prepared from stable SAS-6p–driven Plk4-EGFP that were RNAi treated for the indicated protein for 7 d. (F) Plk4 is phosphorylated. (left) Anti-GFP immunoblots of lysates from control or Slimb-depleted cells transiently expressing inducible Plk4-EGFP. Plk4 accumulates as a doublet (arrowheads) after slimb RNAi. (right) Anti-GFP immunoprecipitates from day 4 Slimb-depleted cells transiently expressing inducible Plk4-EGFP were mock or alkaline phosphatase treated. Plk4 shifts from a broad band (bracket) to a faster migrating single polypeptide (arrowhead). (D–F) Molecular mass is indicated in kilodaltons. (G) Anti-GFP immunoblots showing the levels of transiently expressed SAS-6p–driven Plk4-EGFP and Plk4-SBM–EGFP in 24-h drug-induced cell cycle–arrested cells. Cotransfected Nlp-EGFP was used as a loading control. (H) Anti-GFP immunoblots showing the levels of 4 h–induced Plk4-EGFP and Plk4-SBM–EGFP expression in drug-induced cell cycle–arrested cells. (E and H) α-Tubulin was used as a loading control.

Mentions: We hypothesized that SCFSlimb depletion stabilizes a central regulator of centriole duplication, promoting overduplication. The kinase Plk4 (also called Sak), a tumor suppressor (Ko et al., 2005), is essential for centriole duplication, and Plk4 overexpression produces supernumerary centrioles (Bettencourt-Dias et al., 2005; Habedanck et al., 2005; Kleylein-Sohn et al., 2007; Peel et al., 2007; Rodrigues-Martins et al., 2007). Thus, we explored whether Plk4 is an SCFSlimb target. Consistent with this hypothesis, we identified a potential Slimb-binding site on Drosophila Plk4 downstream of the kinase domain; this sequence (DSGIIT) fits the Slimb-binding consensus (DpSGXXp[S/T]) and is conserved in vertebrate Plk4 orthologues (Fig. 3 A).


The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication.

Rogers GC, Rusan NM, Roberts DM, Peifer M, Rogers SL - J. Cell Biol. (2009)

Plk4 is degraded in a Slimb-dependent manner and is stabilized by perturbing its interaction with Slimb. (A) Plk4 family showing the conserved kinase domain (gray), Polo box motif (striped boxes), and Slimb-binding consensus (black bars). The S293A/T297A SBM should be nondegradable. (B) Preimmune control and anti-Slimb immunoprecipitates from stable S2 cell lysates expressing Plk4-myc were probed for anti-Slimb, SkpA, and myc. (C) Control and anti-GFP immunoprecipitates from S2 cell lysates transiently expressing either inducible wild-type Plk4-EGFP or Plk4-SBM–EGFP were probed for endogenous Slimb. IP, immunoprecipitation. (D) Anti-GFP immunoprecipitates from S2 cell lysates transiently expressing triple Flag-ubiquitin and either inducible wild-type Plk4-EGFP or Plk4-SBM–EGFP were probed with anti-GFP (left) and anti-Flag (right) antibody. IB, immunoblot. (E) Anti-GFP immunoblots of lysates prepared from stable SAS-6p–driven Plk4-EGFP that were RNAi treated for the indicated protein for 7 d. (F) Plk4 is phosphorylated. (left) Anti-GFP immunoblots of lysates from control or Slimb-depleted cells transiently expressing inducible Plk4-EGFP. Plk4 accumulates as a doublet (arrowheads) after slimb RNAi. (right) Anti-GFP immunoprecipitates from day 4 Slimb-depleted cells transiently expressing inducible Plk4-EGFP were mock or alkaline phosphatase treated. Plk4 shifts from a broad band (bracket) to a faster migrating single polypeptide (arrowhead). (D–F) Molecular mass is indicated in kilodaltons. (G) Anti-GFP immunoblots showing the levels of transiently expressed SAS-6p–driven Plk4-EGFP and Plk4-SBM–EGFP in 24-h drug-induced cell cycle–arrested cells. Cotransfected Nlp-EGFP was used as a loading control. (H) Anti-GFP immunoblots showing the levels of 4 h–induced Plk4-EGFP and Plk4-SBM–EGFP expression in drug-induced cell cycle–arrested cells. (E and H) α-Tubulin was used as a loading control.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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fig3: Plk4 is degraded in a Slimb-dependent manner and is stabilized by perturbing its interaction with Slimb. (A) Plk4 family showing the conserved kinase domain (gray), Polo box motif (striped boxes), and Slimb-binding consensus (black bars). The S293A/T297A SBM should be nondegradable. (B) Preimmune control and anti-Slimb immunoprecipitates from stable S2 cell lysates expressing Plk4-myc were probed for anti-Slimb, SkpA, and myc. (C) Control and anti-GFP immunoprecipitates from S2 cell lysates transiently expressing either inducible wild-type Plk4-EGFP or Plk4-SBM–EGFP were probed for endogenous Slimb. IP, immunoprecipitation. (D) Anti-GFP immunoprecipitates from S2 cell lysates transiently expressing triple Flag-ubiquitin and either inducible wild-type Plk4-EGFP or Plk4-SBM–EGFP were probed with anti-GFP (left) and anti-Flag (right) antibody. IB, immunoblot. (E) Anti-GFP immunoblots of lysates prepared from stable SAS-6p–driven Plk4-EGFP that were RNAi treated for the indicated protein for 7 d. (F) Plk4 is phosphorylated. (left) Anti-GFP immunoblots of lysates from control or Slimb-depleted cells transiently expressing inducible Plk4-EGFP. Plk4 accumulates as a doublet (arrowheads) after slimb RNAi. (right) Anti-GFP immunoprecipitates from day 4 Slimb-depleted cells transiently expressing inducible Plk4-EGFP were mock or alkaline phosphatase treated. Plk4 shifts from a broad band (bracket) to a faster migrating single polypeptide (arrowhead). (D–F) Molecular mass is indicated in kilodaltons. (G) Anti-GFP immunoblots showing the levels of transiently expressed SAS-6p–driven Plk4-EGFP and Plk4-SBM–EGFP in 24-h drug-induced cell cycle–arrested cells. Cotransfected Nlp-EGFP was used as a loading control. (H) Anti-GFP immunoblots showing the levels of 4 h–induced Plk4-EGFP and Plk4-SBM–EGFP expression in drug-induced cell cycle–arrested cells. (E and H) α-Tubulin was used as a loading control.
Mentions: We hypothesized that SCFSlimb depletion stabilizes a central regulator of centriole duplication, promoting overduplication. The kinase Plk4 (also called Sak), a tumor suppressor (Ko et al., 2005), is essential for centriole duplication, and Plk4 overexpression produces supernumerary centrioles (Bettencourt-Dias et al., 2005; Habedanck et al., 2005; Kleylein-Sohn et al., 2007; Peel et al., 2007; Rodrigues-Martins et al., 2007). Thus, we explored whether Plk4 is an SCFSlimb target. Consistent with this hypothesis, we identified a potential Slimb-binding site on Drosophila Plk4 downstream of the kinase domain; this sequence (DSGIIT) fits the Slimb-binding consensus (DpSGXXp[S/T]) and is conserved in vertebrate Plk4 orthologues (Fig. 3 A).

Bottom Line: We found that Plk4 binds to Slimb and is an SCF(Slimb) target.Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase.Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
Restricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCF(Slimb) as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCF(Slimb) target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

Show MeSH