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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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The binding modes of Cep192-58mer and Cep152-60mer to Plk4 CPB and mutational analyses of residues critical for the interaction. (a,b) Electrostatic surface representation of the CPB–58mer (a) and the CPB –60mer (b) complexes. The K711 and K/R crater residues of CPB and the E21 of Cep152-60mer are highlighted in rectangles. See Supplementary Figure 3e – h for details. (c–e) Immunoprecipitation (IP) analyses of 293T cells cotransfected with the indicated constructs. –, control vector; numbers, relative signal intensities. (f, g) Characterization of a cancer-associated CEP152 E21K mutation. U2OS cells stably expressing either control vector or siRNA-resistant CEP152-silor CEP152-silE21K mutant were silenced for either control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA) (see Supplementary Table 2), and then immunostained (see Supplementary Figure 4a–c). The numbers of centrosomal Sas6 signals (from 0 to ≥3) among interphase cells (f) and the percentage of mitotic cells with missegregating chromosomes (chr.) (g) were quantified from three independent experiments (≥200 cells/cell line/experiment). Error bars, s.d. Uncropped blot images for c–e are shown in Supplementary Figure 8c–e.
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Figure 3: The binding modes of Cep192-58mer and Cep152-60mer to Plk4 CPB and mutational analyses of residues critical for the interaction. (a,b) Electrostatic surface representation of the CPB–58mer (a) and the CPB –60mer (b) complexes. The K711 and K/R crater residues of CPB and the E21 of Cep152-60mer are highlighted in rectangles. See Supplementary Figure 3e – h for details. (c–e) Immunoprecipitation (IP) analyses of 293T cells cotransfected with the indicated constructs. –, control vector; numbers, relative signal intensities. (f, g) Characterization of a cancer-associated CEP152 E21K mutation. U2OS cells stably expressing either control vector or siRNA-resistant CEP152-silor CEP152-silE21K mutant were silenced for either control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA) (see Supplementary Table 2), and then immunostained (see Supplementary Figure 4a–c). The numbers of centrosomal Sas6 signals (from 0 to ≥3) among interphase cells (f) and the percentage of mitotic cells with missegregating chromosomes (chr.) (g) were quantified from three independent experiments (≥200 cells/cell line/experiment). Error bars, s.d. Uncropped blot images for c–e are shown in Supplementary Figure 8c–e.

Mentions: Cep192-58mer and Cep152-60mer formed three major contacts with CPB. Although the centrally located α-helical sequences from the 58mer and the 60mer exhibit no detectable sequence homology (Fig. 2c), they bound to an overlapped region of CPB by engaging in multiple hydrophobic and hydrogen bond interactions with several common residues along the α1 and β1 of PB1 (Fig. 3a,b, left; see details in Supplementary Fig. 3e). The 58mer α-helix was skewed at an oblique angle to the axis of the CPB α1, whereas the 60mer α-helix was antiparallel to the CPB α1, forming tightly packed hydrophobic interactions between the two α-helices. The coiled-coil interface was reinforced by salt bridges between the D20 and D23 of the 60mer and the K711 (rectangled) of the CPB (Fig. 3b, left).


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)

The binding modes of Cep192-58mer and Cep152-60mer to Plk4 CPB and mutational analyses of residues critical for the interaction. (a,b) Electrostatic surface representation of the CPB–58mer (a) and the CPB –60mer (b) complexes. The K711 and K/R crater residues of CPB and the E21 of Cep152-60mer are highlighted in rectangles. See Supplementary Figure 3e – h for details. (c–e) Immunoprecipitation (IP) analyses of 293T cells cotransfected with the indicated constructs. –, control vector; numbers, relative signal intensities. (f, g) Characterization of a cancer-associated CEP152 E21K mutation. U2OS cells stably expressing either control vector or siRNA-resistant CEP152-silor CEP152-silE21K mutant were silenced for either control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA) (see Supplementary Table 2), and then immunostained (see Supplementary Figure 4a–c). The numbers of centrosomal Sas6 signals (from 0 to ≥3) among interphase cells (f) and the percentage of mitotic cells with missegregating chromosomes (chr.) (g) were quantified from three independent experiments (≥200 cells/cell line/experiment). Error bars, s.d. Uncropped blot images for c–e are shown in Supplementary Figure 8c–e.
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Figure 3: The binding modes of Cep192-58mer and Cep152-60mer to Plk4 CPB and mutational analyses of residues critical for the interaction. (a,b) Electrostatic surface representation of the CPB–58mer (a) and the CPB –60mer (b) complexes. The K711 and K/R crater residues of CPB and the E21 of Cep152-60mer are highlighted in rectangles. See Supplementary Figure 3e – h for details. (c–e) Immunoprecipitation (IP) analyses of 293T cells cotransfected with the indicated constructs. –, control vector; numbers, relative signal intensities. (f, g) Characterization of a cancer-associated CEP152 E21K mutation. U2OS cells stably expressing either control vector or siRNA-resistant CEP152-silor CEP152-silE21K mutant were silenced for either control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA) (see Supplementary Table 2), and then immunostained (see Supplementary Figure 4a–c). The numbers of centrosomal Sas6 signals (from 0 to ≥3) among interphase cells (f) and the percentage of mitotic cells with missegregating chromosomes (chr.) (g) were quantified from three independent experiments (≥200 cells/cell line/experiment). Error bars, s.d. Uncropped blot images for c–e are shown in Supplementary Figure 8c–e.
Mentions: Cep192-58mer and Cep152-60mer formed three major contacts with CPB. Although the centrally located α-helical sequences from the 58mer and the 60mer exhibit no detectable sequence homology (Fig. 2c), they bound to an overlapped region of CPB by engaging in multiple hydrophobic and hydrogen bond interactions with several common residues along the α1 and β1 of PB1 (Fig. 3a,b, left; see details in Supplementary Fig. 3e). The 58mer α-helix was skewed at an oblique angle to the axis of the CPB α1, whereas the 60mer α-helix was antiparallel to the CPB α1, forming tightly packed hydrophobic interactions between the two α-helices. The coiled-coil interface was reinforced by salt bridges between the D20 and D23 of the 60mer and the K711 (rectangled) of the CPB (Fig. 3b, left).

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