Limits...
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.

Show MeSH

Related in: MedlinePlus

Two distinct sizes of Plk4 ring structures in the absence or presence of the Cep152 ring around Cep192-decorated centrioles. (a) 3D -SIM images showing asynchronously growing U2OS cells co-immunostained with anti-Plk4 (red), anti-Sas6 (blue; pseudo-colored in gray), Alexa 647 (magenta)-conjugated anti-Cep192 N-terminal (N), and Alexa 488 (green)-conjugated anti-Cep152 middle region (M) antibodies. Arrows in the 1st panel indicate the outer diameters of two Plk4 rings—one from a daughter (D) and the other from a mother (M) centriole before and after Cep152 recruitment, respectively. A bracket on the 2ndpanel indicates Plk4 signals colocalized with a nascent Cep152 toroid assembling at a daughter centriole. Arrowheads on the 3rd and 4th panels indicate dot -like Plk4 (red) signals colocalized with Sas6 (gray) on Cep152 toroids. Scale bars, 0.5 μm. (b) Quantification of the outer diameters of Cep192, Cep152, and Plk4 ring signals for the samples in Figure 1a. G1 centrioles prior to Cep152 recruitment (n=25) or after Cep152 recruitment (n=69), or S centrioles without discernable Plk4 ring signals (n=41) were measured. Error bars, s.d. (c) An immunoblot showing U2OS cells silenced for control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA). Asterisk, cross-reacting protein. (d,e) Immunostaining (d) of the cells in c and subsequent quantification (e) from three independent experiments (n=105 for GL siRNA, n=116 for CEP152 siRNA). Arrows in d, the outer diameters of Cep192 and Plk4 rings; scale bars in d, 0.5 μm; error bars in e, s.d. An uncropped blot image for c is shown in Supplementary Figure 8a.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4125498&req=5

Figure 1: Two distinct sizes of Plk4 ring structures in the absence or presence of the Cep152 ring around Cep192-decorated centrioles. (a) 3D -SIM images showing asynchronously growing U2OS cells co-immunostained with anti-Plk4 (red), anti-Sas6 (blue; pseudo-colored in gray), Alexa 647 (magenta)-conjugated anti-Cep192 N-terminal (N), and Alexa 488 (green)-conjugated anti-Cep152 middle region (M) antibodies. Arrows in the 1st panel indicate the outer diameters of two Plk4 rings—one from a daughter (D) and the other from a mother (M) centriole before and after Cep152 recruitment, respectively. A bracket on the 2ndpanel indicates Plk4 signals colocalized with a nascent Cep152 toroid assembling at a daughter centriole. Arrowheads on the 3rd and 4th panels indicate dot -like Plk4 (red) signals colocalized with Sas6 (gray) on Cep152 toroids. Scale bars, 0.5 μm. (b) Quantification of the outer diameters of Cep192, Cep152, and Plk4 ring signals for the samples in Figure 1a. G1 centrioles prior to Cep152 recruitment (n=25) or after Cep152 recruitment (n=69), or S centrioles without discernable Plk4 ring signals (n=41) were measured. Error bars, s.d. (c) An immunoblot showing U2OS cells silenced for control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA). Asterisk, cross-reacting protein. (d,e) Immunostaining (d) of the cells in c and subsequent quantification (e) from three independent experiments (n=105 for GL siRNA, n=116 for CEP152 siRNA). Arrows in d, the outer diameters of Cep192 and Plk4 rings; scale bars in d, 0.5 μm; error bars in e, s.d. An uncropped blot image for c is shown in Supplementary Figure 8a.

Mentions: To understand the functional relationships between Plk4 and the two Plk4-binding scaffold proteins, Cep192 and Cep152, at the initial stage of centriole biogenesis, we first examined the subcentriolar localization patterns of these proteins in U2OS cells by performing three-dimensional structured illumination microscopy (3D-SIM) analysis (Fig. 1 and Supplementary Fig. 1).


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)

Two distinct sizes of Plk4 ring structures in the absence or presence of the Cep152 ring around Cep192-decorated centrioles. (a) 3D -SIM images showing asynchronously growing U2OS cells co-immunostained with anti-Plk4 (red), anti-Sas6 (blue; pseudo-colored in gray), Alexa 647 (magenta)-conjugated anti-Cep192 N-terminal (N), and Alexa 488 (green)-conjugated anti-Cep152 middle region (M) antibodies. Arrows in the 1st panel indicate the outer diameters of two Plk4 rings—one from a daughter (D) and the other from a mother (M) centriole before and after Cep152 recruitment, respectively. A bracket on the 2ndpanel indicates Plk4 signals colocalized with a nascent Cep152 toroid assembling at a daughter centriole. Arrowheads on the 3rd and 4th panels indicate dot -like Plk4 (red) signals colocalized with Sas6 (gray) on Cep152 toroids. Scale bars, 0.5 μm. (b) Quantification of the outer diameters of Cep192, Cep152, and Plk4 ring signals for the samples in Figure 1a. G1 centrioles prior to Cep152 recruitment (n=25) or after Cep152 recruitment (n=69), or S centrioles without discernable Plk4 ring signals (n=41) were measured. Error bars, s.d. (c) An immunoblot showing U2OS cells silenced for control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA). Asterisk, cross-reacting protein. (d,e) Immunostaining (d) of the cells in c and subsequent quantification (e) from three independent experiments (n=105 for GL siRNA, n=116 for CEP152 siRNA). Arrows in d, the outer diameters of Cep192 and Plk4 rings; scale bars in d, 0.5 μm; error bars in e, s.d. An uncropped blot image for c is shown in Supplementary Figure 8a.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Two distinct sizes of Plk4 ring structures in the absence or presence of the Cep152 ring around Cep192-decorated centrioles. (a) 3D -SIM images showing asynchronously growing U2OS cells co-immunostained with anti-Plk4 (red), anti-Sas6 (blue; pseudo-colored in gray), Alexa 647 (magenta)-conjugated anti-Cep192 N-terminal (N), and Alexa 488 (green)-conjugated anti-Cep152 middle region (M) antibodies. Arrows in the 1st panel indicate the outer diameters of two Plk4 rings—one from a daughter (D) and the other from a mother (M) centriole before and after Cep152 recruitment, respectively. A bracket on the 2ndpanel indicates Plk4 signals colocalized with a nascent Cep152 toroid assembling at a daughter centriole. Arrowheads on the 3rd and 4th panels indicate dot -like Plk4 (red) signals colocalized with Sas6 (gray) on Cep152 toroids. Scale bars, 0.5 μm. (b) Quantification of the outer diameters of Cep192, Cep152, and Plk4 ring signals for the samples in Figure 1a. G1 centrioles prior to Cep152 recruitment (n=25) or after Cep152 recruitment (n=69), or S centrioles without discernable Plk4 ring signals (n=41) were measured. Error bars, s.d. (c) An immunoblot showing U2OS cells silenced for control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA). Asterisk, cross-reacting protein. (d,e) Immunostaining (d) of the cells in c and subsequent quantification (e) from three independent experiments (n=105 for GL siRNA, n=116 for CEP152 siRNA). Arrows in d, the outer diameters of Cep192 and Plk4 rings; scale bars in d, 0.5 μm; error bars in e, s.d. An uncropped blot image for c is shown in Supplementary Figure 8a.
Mentions: To understand the functional relationships between Plk4 and the two Plk4-binding scaffold proteins, Cep192 and Cep152, at the initial stage of centriole biogenesis, we first examined the subcentriolar localization patterns of these proteins in U2OS cells by performing three-dimensional structured illumination microscopy (3D-SIM) analysis (Fig. 1 and Supplementary Fig. 1).

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