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Molecular basis for unidirectional scaffold switching of human Plk4 in centriole biogenesis.

Park SY, Park JE, Kim TS, Kim JH, Kwak MJ, Ku B, Tian L, Murugan RN, Ahn M, Komiya S, Hojo H, Kim NH, Kim BY, Bang JK, Erikson RL, Lee KW, Kim SJ, Oh BH, Yang W, Lee KS - Nat. Struct. Mol. Biol. (2014)

Bottom Line: Crystal-structure analyses revealed that Cep192- and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner.The Cep152 peptide bound to the CPB markedly better than did the Cep192 peptide and effectively 'snatched' the CPB away from a preformed CPB-Cep192 peptide complex.A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. [2] Advanced Institutes of Convergence Technology, Seoul National University, Suwon, Republic of Korea. [3].

ABSTRACT
Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. We show that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal-structure analyses revealed that Cep192- and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152 peptide bound to the CPB markedly better than did the Cep192 peptide and effectively 'snatched' the CPB away from a preformed CPB-Cep192 peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.

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Biochemical and structural analysis of Plk4 CPB in complex with Cep192-58mer or Cep152-60mer. (a) Interaction of Plk4 with Cep192 and Cep152 at endogenous levels. HeLa cell lysates were subjected to immunoprecipitation (IP) analysis with the indicated antibodies cross-linked to agarose beads. GL siRNA, Luciferase siRNA; IgG, control rabbit IgG. (b) Immunoprecipitation (IP) analysis was performed with 293T cells cotransfected with the indicated constructs. White asterisks, remaining FLAG-Plk4 CPB signals from the second panel immunoblot; Black asterisks, non-specific bands from IgG light chain. (c) Reverse alignment of Cep152-60mer and Cep192-58mer. Identical (vertical line) and conserved (colon) residues are indicated. (d) MBP pull-downs were performed using purified proteins. CBB, Coomassie-stained gel; asterisks, contaminated proteins; numbers, the relative amount of Cep192-58mer or Cep152-60mer bound to a CPB dimer (2.0). (e) Immunoprecipitation (IP) analysis with 293T cells cotransfected with the indicated constructs. To achieve stable overexpression, a kinase-inactive Plk4 K41M mutant was used. CBB, Coomassie-stained gel; arrows, coimmunoprecipitated FLAG-Plk4; asterisk, cross-reacting protein; H.C. and L.C., IgG heavy and light chains. (f,g) Overall structures of the CPB–Cep192-58mer and the CPB–Cep152-60mer complexes. Each subunit (blue or orange) of a homodimeric CPB consists of PB1 and PB2 folds. N, N-terminus; C, C-terminus; D-rich, D-rich motif. Uncropped blot images for a and b are shown in Supplementary Figure 8b,c.
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Figure 2: Biochemical and structural analysis of Plk4 CPB in complex with Cep192-58mer or Cep152-60mer. (a) Interaction of Plk4 with Cep192 and Cep152 at endogenous levels. HeLa cell lysates were subjected to immunoprecipitation (IP) analysis with the indicated antibodies cross-linked to agarose beads. GL siRNA, Luciferase siRNA; IgG, control rabbit IgG. (b) Immunoprecipitation (IP) analysis was performed with 293T cells cotransfected with the indicated constructs. White asterisks, remaining FLAG-Plk4 CPB signals from the second panel immunoblot; Black asterisks, non-specific bands from IgG light chain. (c) Reverse alignment of Cep152-60mer and Cep192-58mer. Identical (vertical line) and conserved (colon) residues are indicated. (d) MBP pull-downs were performed using purified proteins. CBB, Coomassie-stained gel; asterisks, contaminated proteins; numbers, the relative amount of Cep192-58mer or Cep152-60mer bound to a CPB dimer (2.0). (e) Immunoprecipitation (IP) analysis with 293T cells cotransfected with the indicated constructs. To achieve stable overexpression, a kinase-inactive Plk4 K41M mutant was used. CBB, Coomassie-stained gel; arrows, coimmunoprecipitated FLAG-Plk4; asterisk, cross-reacting protein; H.C. and L.C., IgG heavy and light chains. (f,g) Overall structures of the CPB–Cep192-58mer and the CPB–Cep152-60mer complexes. Each subunit (blue or orange) of a homodimeric CPB consists of PB1 and PB2 folds. N, N-terminus; C, C-terminus; D-rich, D-rich motif. Uncropped blot images for a and b are shown in Supplementary Figure 8b,c.

Mentions: Since Plk4 colocalized with Cep192 and Cep152 at subcentriolar structures (Fig. 1), we examined whether it interacts with these proteins in vivo. We observed that immunoprecipitation of Cep192 or Cep152 coprecipitated Plk4 at the endogenous level (Fig. 2a). However, Cep192 and Cep152 did not interact with each other under the same conditions (Fig. 2a). These findings suggest that Plk4 forms two distinct complexes with Cep192 or Cep152.


Molecular basis for unidirectional scaffold switching of human Plk4 in centriole biogenesis.

Park SY, Park JE, Kim TS, Kim JH, Kwak MJ, Ku B, Tian L, Murugan RN, Ahn M, Komiya S, Hojo H, Kim NH, Kim BY, Bang JK, Erikson RL, Lee KW, Kim SJ, Oh BH, Yang W, Lee KS - Nat. Struct. Mol. Biol. (2014)

Biochemical and structural analysis of Plk4 CPB in complex with Cep192-58mer or Cep152-60mer. (a) Interaction of Plk4 with Cep192 and Cep152 at endogenous levels. HeLa cell lysates were subjected to immunoprecipitation (IP) analysis with the indicated antibodies cross-linked to agarose beads. GL siRNA, Luciferase siRNA; IgG, control rabbit IgG. (b) Immunoprecipitation (IP) analysis was performed with 293T cells cotransfected with the indicated constructs. White asterisks, remaining FLAG-Plk4 CPB signals from the second panel immunoblot; Black asterisks, non-specific bands from IgG light chain. (c) Reverse alignment of Cep152-60mer and Cep192-58mer. Identical (vertical line) and conserved (colon) residues are indicated. (d) MBP pull-downs were performed using purified proteins. CBB, Coomassie-stained gel; asterisks, contaminated proteins; numbers, the relative amount of Cep192-58mer or Cep152-60mer bound to a CPB dimer (2.0). (e) Immunoprecipitation (IP) analysis with 293T cells cotransfected with the indicated constructs. To achieve stable overexpression, a kinase-inactive Plk4 K41M mutant was used. CBB, Coomassie-stained gel; arrows, coimmunoprecipitated FLAG-Plk4; asterisk, cross-reacting protein; H.C. and L.C., IgG heavy and light chains. (f,g) Overall structures of the CPB–Cep192-58mer and the CPB–Cep152-60mer complexes. Each subunit (blue or orange) of a homodimeric CPB consists of PB1 and PB2 folds. N, N-terminus; C, C-terminus; D-rich, D-rich motif. Uncropped blot images for a and b are shown in Supplementary Figure 8b,c.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4125498&req=5

Figure 2: Biochemical and structural analysis of Plk4 CPB in complex with Cep192-58mer or Cep152-60mer. (a) Interaction of Plk4 with Cep192 and Cep152 at endogenous levels. HeLa cell lysates were subjected to immunoprecipitation (IP) analysis with the indicated antibodies cross-linked to agarose beads. GL siRNA, Luciferase siRNA; IgG, control rabbit IgG. (b) Immunoprecipitation (IP) analysis was performed with 293T cells cotransfected with the indicated constructs. White asterisks, remaining FLAG-Plk4 CPB signals from the second panel immunoblot; Black asterisks, non-specific bands from IgG light chain. (c) Reverse alignment of Cep152-60mer and Cep192-58mer. Identical (vertical line) and conserved (colon) residues are indicated. (d) MBP pull-downs were performed using purified proteins. CBB, Coomassie-stained gel; asterisks, contaminated proteins; numbers, the relative amount of Cep192-58mer or Cep152-60mer bound to a CPB dimer (2.0). (e) Immunoprecipitation (IP) analysis with 293T cells cotransfected with the indicated constructs. To achieve stable overexpression, a kinase-inactive Plk4 K41M mutant was used. CBB, Coomassie-stained gel; arrows, coimmunoprecipitated FLAG-Plk4; asterisk, cross-reacting protein; H.C. and L.C., IgG heavy and light chains. (f,g) Overall structures of the CPB–Cep192-58mer and the CPB–Cep152-60mer complexes. Each subunit (blue or orange) of a homodimeric CPB consists of PB1 and PB2 folds. N, N-terminus; C, C-terminus; D-rich, D-rich motif. Uncropped blot images for a and b are shown in Supplementary Figure 8b,c.
Mentions: Since Plk4 colocalized with Cep192 and Cep152 at subcentriolar structures (Fig. 1), we examined whether it interacts with these proteins in vivo. We observed that immunoprecipitation of Cep192 or Cep152 coprecipitated Plk4 at the endogenous level (Fig. 2a). However, Cep192 and Cep152 did not interact with each other under the same conditions (Fig. 2a). These findings suggest that Plk4 forms two distinct complexes with Cep192 or Cep152.

Bottom Line: Crystal-structure analyses revealed that Cep192- and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner.The Cep152 peptide bound to the CPB markedly better than did the Cep192 peptide and effectively 'snatched' the CPB away from a preformed CPB-Cep192 peptide complex.A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. [2] Advanced Institutes of Convergence Technology, Seoul National University, Suwon, Republic of Korea. [3].

ABSTRACT
Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. We show that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal-structure analyses revealed that Cep192- and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152 peptide bound to the CPB markedly better than did the Cep192 peptide and effectively 'snatched' the CPB away from a preformed CPB-Cep192 peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.

Show MeSH
Related in: MedlinePlus