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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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Mechanistic basis of repositioning Cep192-bound Plk4 with incoming Cep152 at daughter centrioles. (a) Superposition of the structures of Cep192-58mer and Cep152-60mer individually bound to Plk4 CPB. Arrowed bracket indicates the region of steric clash between the 60mer and the 58mer binding to two different subunits of a CPB dimer. N, N-terminus; C, C-terminus. (b) Fluorescence polarization (FP)-based binding assays were performed using Alexa 488 -conjugated (*) MBP or MBP -fused ligand and increasing concentrations of unconjugated Plk4 CPB. (c) FP inhibition assays were performed by providing increasing concentrations of MBP -Cep152-60mer or MBP-Cep192-58mer to preformed MBP-Cep192-58mer*–CPB complex or MBP-Cep152-60mer*–CPB complex, respectively. The data obtained from three independent experiments. Error bars, s.d. (d) A biochemical assay demonstrating the ability of Cep152-60mer to snatch CPB from the Cep192-58mer–CPB complex. Proteins eluted after the addition of increasing concentrations of MBP-Cep152-60mer were analyzed. See detailed procedures in Methods. Slanted arrows, the first fraction containing the Coomassie-stainable amount of co-eluted MBP-Cep152-60mer and CPB; Vertical arrows, the fraction with the highest levels of MBP-Cep152-60mer and CPB; Asterisk, a non-specific protein co-migrating with the 60mer; Numbers, relative amounts of CPB coeluted with the 60mer.
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Figure 4: Mechanistic basis of repositioning Cep192-bound Plk4 with incoming Cep152 at daughter centrioles. (a) Superposition of the structures of Cep192-58mer and Cep152-60mer individually bound to Plk4 CPB. Arrowed bracket indicates the region of steric clash between the 60mer and the 58mer binding to two different subunits of a CPB dimer. N, N-terminus; C, C-terminus. (b) Fluorescence polarization (FP)-based binding assays were performed using Alexa 488 -conjugated (*) MBP or MBP -fused ligand and increasing concentrations of unconjugated Plk4 CPB. (c) FP inhibition assays were performed by providing increasing concentrations of MBP -Cep152-60mer or MBP-Cep192-58mer to preformed MBP-Cep192-58mer*–CPB complex or MBP-Cep152-60mer*–CPB complex, respectively. The data obtained from three independent experiments. Error bars, s.d. (d) A biochemical assay demonstrating the ability of Cep152-60mer to snatch CPB from the Cep192-58mer–CPB complex. Proteins eluted after the addition of increasing concentrations of MBP-Cep152-60mer were analyzed. See detailed procedures in Methods. Slanted arrows, the first fraction containing the Coomassie-stainable amount of co-eluted MBP-Cep152-60mer and CPB; Vertical arrows, the fraction with the highest levels of MBP-Cep152-60mer and CPB; Asterisk, a non-specific protein co-migrating with the 60mer; Numbers, relative amounts of CPB coeluted with the 60mer.

Mentions: Discrete enlargement of Plk4 ring structures on Cep152-recruited centrioles (Fig. 1a) and similar binding modes of Cep192-58mer and Cep152-60mer to CPB (Fig. 2f,g) suggest that Cep192–Plk4 and Cep152–Plk4 complexes may not coexist in vivo. Indeed, superposition of CPB-bound Cep192-58mer and Cep152-60mer (root-mean-square deviation [r.m.s.d.] of 2.2 Å across all Cα atoms of the CPB dimer) showed that Cep152 -60mer not only directly interfered with the binding of Cep192-58mer to the same CPB subunit, but also clashed with the second Cep192-58mer already bound to the other CPB subunit (Fig. 4a), suggesting that binding of Cep192-58mer and Cep152-60mer to CPB is mutually exclusive.


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)

Mechanistic basis of repositioning Cep192-bound Plk4 with incoming Cep152 at daughter centrioles. (a) Superposition of the structures of Cep192-58mer and Cep152-60mer individually bound to Plk4 CPB. Arrowed bracket indicates the region of steric clash between the 60mer and the 58mer binding to two different subunits of a CPB dimer. N, N-terminus; C, C-terminus. (b) Fluorescence polarization (FP)-based binding assays were performed using Alexa 488 -conjugated (*) MBP or MBP -fused ligand and increasing concentrations of unconjugated Plk4 CPB. (c) FP inhibition assays were performed by providing increasing concentrations of MBP -Cep152-60mer or MBP-Cep192-58mer to preformed MBP-Cep192-58mer*–CPB complex or MBP-Cep152-60mer*–CPB complex, respectively. The data obtained from three independent experiments. Error bars, s.d. (d) A biochemical assay demonstrating the ability of Cep152-60mer to snatch CPB from the Cep192-58mer–CPB complex. Proteins eluted after the addition of increasing concentrations of MBP-Cep152-60mer were analyzed. See detailed procedures in Methods. Slanted arrows, the first fraction containing the Coomassie-stainable amount of co-eluted MBP-Cep152-60mer and CPB; Vertical arrows, the fraction with the highest levels of MBP-Cep152-60mer and CPB; Asterisk, a non-specific protein co-migrating with the 60mer; Numbers, relative amounts of CPB coeluted with the 60mer.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Mechanistic basis of repositioning Cep192-bound Plk4 with incoming Cep152 at daughter centrioles. (a) Superposition of the structures of Cep192-58mer and Cep152-60mer individually bound to Plk4 CPB. Arrowed bracket indicates the region of steric clash between the 60mer and the 58mer binding to two different subunits of a CPB dimer. N, N-terminus; C, C-terminus. (b) Fluorescence polarization (FP)-based binding assays were performed using Alexa 488 -conjugated (*) MBP or MBP -fused ligand and increasing concentrations of unconjugated Plk4 CPB. (c) FP inhibition assays were performed by providing increasing concentrations of MBP -Cep152-60mer or MBP-Cep192-58mer to preformed MBP-Cep192-58mer*–CPB complex or MBP-Cep152-60mer*–CPB complex, respectively. The data obtained from three independent experiments. Error bars, s.d. (d) A biochemical assay demonstrating the ability of Cep152-60mer to snatch CPB from the Cep192-58mer–CPB complex. Proteins eluted after the addition of increasing concentrations of MBP-Cep152-60mer were analyzed. See detailed procedures in Methods. Slanted arrows, the first fraction containing the Coomassie-stainable amount of co-eluted MBP-Cep152-60mer and CPB; Vertical arrows, the fraction with the highest levels of MBP-Cep152-60mer and CPB; Asterisk, a non-specific protein co-migrating with the 60mer; Numbers, relative amounts of CPB coeluted with the 60mer.
Mentions: Discrete enlargement of Plk4 ring structures on Cep152-recruited centrioles (Fig. 1a) and similar binding modes of Cep192-58mer and Cep152-60mer to CPB (Fig. 2f,g) suggest that Cep192–Plk4 and Cep152–Plk4 complexes may not coexist in vivo. Indeed, superposition of CPB-bound Cep192-58mer and Cep152-60mer (root-mean-square deviation [r.m.s.d.] of 2.2 Å across all Cα atoms of the CPB dimer) showed that Cep152 -60mer not only directly interfered with the binding of Cep192-58mer to the same CPB subunit, but also clashed with the second Cep192-58mer already bound to the other CPB subunit (Fig. 4a), suggesting that binding of Cep192-58mer and Cep152-60mer to CPB is mutually exclusive.

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