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Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes.

Platani M, Trinkle-Mulcahy L, Porter M, Jeyaprakash AA, Earnshaw WC - J. Cell Biol. (2015)

Bottom Line: In this study, we report that Mio, a highly conserved member of the SEACAT/GATOR2 complex necessary for the activation of mTORC1 kinase, plays a critical role in mitotic spindle formation and subsequent chromosome segregation by regulating the proper concentration of active key mitotic kinases Plk1 and Aurora A at centrosomes and spindle poles.Mio-depleted cells showed reduced activation of Plk1 and Aurora A kinase at spindle poles and an impaired localization of MCAK and HURP, two key regulators of mitotic spindle formation and known substrates of Aurora A kinase, resulting in spindle assembly and cytokinesis defects.Our results indicate that a major function of Mio in mitosis is to regulate the activation/deactivation of Plk1 and Aurora A, possibly by linking them to mTOR signaling in a pathway to promote faithful mitotic progression.

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Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK m.platani@ed.ac.uk Bill.Earnshaw@ed.ac.uk.

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Mitotic progression is impaired in the absence of the Ring/PHD domain. (A) Schematic diagram showing the domain structure of Mio and the sequence of its C-terminal Ring/PHD domain. The predicted cysteines and histidines involved in zinc coordination are labeled in red. (B) Cartoon representation of predicted 3D molecular structure of Mio C-terminal Ring/PHD domain showing the mutated residues in stick form. Zinc ions are shown in ball representation (as white spheres). (C) Mitotic progression scatter plots with NEBD as T = 0 in control (blue), Mio siRNA-treated cells with and without (red) expression of siRNA-resistant versions of wild-type GFP-Mio (green), GFP-MioRing8A (black), and GFP-MioΔPHD (purple) from live cell videos. n = 214 cells from two independent experiments. Statistical significance was determined by an unpaired Student’s t test.
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fig8: Mitotic progression is impaired in the absence of the Ring/PHD domain. (A) Schematic diagram showing the domain structure of Mio and the sequence of its C-terminal Ring/PHD domain. The predicted cysteines and histidines involved in zinc coordination are labeled in red. (B) Cartoon representation of predicted 3D molecular structure of Mio C-terminal Ring/PHD domain showing the mutated residues in stick form. Zinc ions are shown in ball representation (as white spheres). (C) Mitotic progression scatter plots with NEBD as T = 0 in control (blue), Mio siRNA-treated cells with and without (red) expression of siRNA-resistant versions of wild-type GFP-Mio (green), GFP-MioRing8A (black), and GFP-MioΔPHD (purple) from live cell videos. n = 214 cells from two independent experiments. Statistical significance was determined by an unpaired Student’s t test.

Mentions: Mio is highly conserved from yeast to mammals (Iida and Lilly, 2004). Its N-terminal domain contains several WD40 repeats that are likely to be involved in protein–protein interaction (Smith et al., 1999). Mio also has a C-terminal domain with a pattern of conserved histidine and cysteine residues resembling a RING/PHD domain (Fig. 8 A). Such RING/PHD domains frequently mediate E3 ligase activity (Deshaies and Joazeiro, 2009). We used the Protein Homology/Analogy Recognition Engine version 2.0 (Phyre2) server (Kelley and Sternberg, 2009) to model Mio’s RING/PHD domain structure on the crystal structure of Ring E3 ubiquitin ligase RNF8 bound to a UBC13/MMS2 heterodimer (Protein Data Bank accession no. 4ORH). Cysteine and histidine residues that are predicted to coordinate the two zinc atoms and help maintain the domain structure are highlighted (Fig. 8, A [red] and B [labels]). Based on this model, we decided to create two Mio mutants: a C-terminal RING/PHD deletion (ΔPHD) missing the last 91 amino acids and a combination of eight specific point mutations (C785, C788, C830, H832, H835, H838, C849, and C854) that convert predicted zinc-binding cysteine and histidine residues to alanine. This would be predicted to disrupt the folding of the Ring domain (Fig. 8 B).


Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes.

Platani M, Trinkle-Mulcahy L, Porter M, Jeyaprakash AA, Earnshaw WC - J. Cell Biol. (2015)

Mitotic progression is impaired in the absence of the Ring/PHD domain. (A) Schematic diagram showing the domain structure of Mio and the sequence of its C-terminal Ring/PHD domain. The predicted cysteines and histidines involved in zinc coordination are labeled in red. (B) Cartoon representation of predicted 3D molecular structure of Mio C-terminal Ring/PHD domain showing the mutated residues in stick form. Zinc ions are shown in ball representation (as white spheres). (C) Mitotic progression scatter plots with NEBD as T = 0 in control (blue), Mio siRNA-treated cells with and without (red) expression of siRNA-resistant versions of wild-type GFP-Mio (green), GFP-MioRing8A (black), and GFP-MioΔPHD (purple) from live cell videos. n = 214 cells from two independent experiments. Statistical significance was determined by an unpaired Student’s t test.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494011&req=5

fig8: Mitotic progression is impaired in the absence of the Ring/PHD domain. (A) Schematic diagram showing the domain structure of Mio and the sequence of its C-terminal Ring/PHD domain. The predicted cysteines and histidines involved in zinc coordination are labeled in red. (B) Cartoon representation of predicted 3D molecular structure of Mio C-terminal Ring/PHD domain showing the mutated residues in stick form. Zinc ions are shown in ball representation (as white spheres). (C) Mitotic progression scatter plots with NEBD as T = 0 in control (blue), Mio siRNA-treated cells with and without (red) expression of siRNA-resistant versions of wild-type GFP-Mio (green), GFP-MioRing8A (black), and GFP-MioΔPHD (purple) from live cell videos. n = 214 cells from two independent experiments. Statistical significance was determined by an unpaired Student’s t test.
Mentions: Mio is highly conserved from yeast to mammals (Iida and Lilly, 2004). Its N-terminal domain contains several WD40 repeats that are likely to be involved in protein–protein interaction (Smith et al., 1999). Mio also has a C-terminal domain with a pattern of conserved histidine and cysteine residues resembling a RING/PHD domain (Fig. 8 A). Such RING/PHD domains frequently mediate E3 ligase activity (Deshaies and Joazeiro, 2009). We used the Protein Homology/Analogy Recognition Engine version 2.0 (Phyre2) server (Kelley and Sternberg, 2009) to model Mio’s RING/PHD domain structure on the crystal structure of Ring E3 ubiquitin ligase RNF8 bound to a UBC13/MMS2 heterodimer (Protein Data Bank accession no. 4ORH). Cysteine and histidine residues that are predicted to coordinate the two zinc atoms and help maintain the domain structure are highlighted (Fig. 8, A [red] and B [labels]). Based on this model, we decided to create two Mio mutants: a C-terminal RING/PHD deletion (ΔPHD) missing the last 91 amino acids and a combination of eight specific point mutations (C785, C788, C830, H832, H835, H838, C849, and C854) that convert predicted zinc-binding cysteine and histidine residues to alanine. This would be predicted to disrupt the folding of the Ring domain (Fig. 8 B).

Bottom Line: In this study, we report that Mio, a highly conserved member of the SEACAT/GATOR2 complex necessary for the activation of mTORC1 kinase, plays a critical role in mitotic spindle formation and subsequent chromosome segregation by regulating the proper concentration of active key mitotic kinases Plk1 and Aurora A at centrosomes and spindle poles.Mio-depleted cells showed reduced activation of Plk1 and Aurora A kinase at spindle poles and an impaired localization of MCAK and HURP, two key regulators of mitotic spindle formation and known substrates of Aurora A kinase, resulting in spindle assembly and cytokinesis defects.Our results indicate that a major function of Mio in mitosis is to regulate the activation/deactivation of Plk1 and Aurora A, possibly by linking them to mTOR signaling in a pathway to promote faithful mitotic progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK m.platani@ed.ac.uk Bill.Earnshaw@ed.ac.uk.

Show MeSH
Related in: MedlinePlus