Cyclin A/Cdk1 modulates Plk1 activity in prometaphase to regulate kinetochore-microtubule attachment stability
- Geisel School of Medicine at Dartmouth, United States
- Norris Cotton Cancer Center, United States
Figures
Figure 1 with 3 supplements
Identification of Cyclin A/Cdk1 substrates in prometaphase cells using SILAC-MS/MS.
(A) Experimental design for SILAC approach to generate two independently labeled prometaphase cell populations with differential Cyclin A levels for analysis of phosphorylation changes. (B) Western …
Figure 1—figure supplement 1
Triaged Cyclin A/Cdk1 Prometaphase Substrates by Category.
Cartoon spindle depicting examples of Cdk1 substrates that were preferentially phosphorylated in the presence of Cyclin A. Substrates are separated by mitotic function. Color intensity represents …
Figure 1—figure supplement 2
Triaged Aurora B Prometaphase Substrates by Category.
Cartoon spindle depicting examples of Aurora B kinase substrates that were preferentially phosphorylated in the presence of Cyclin A. Substrates are separated by mitotic function. Color intensity …
Figure 1—figure supplement 3
Triaged Plk1 Prometaphase Substrates by Category.
Cartoon spindle depicting examples of Plk1 substrates that were preferentially phosphorylated in the presence of Cyclin A. Substrates are separated by mitotic function. Color intensity represents …
Figure 2
MYPT1 is a mitotic Cyclin A/Cdk1 substrate.
(A) Chromosome spreads from nocodazole-treated U2OS before (CT) and after si-RNA-mediated Cyclin A-knockdown (CycA KD) stained for DNA, centromere-specific human antisera (ACA), antibody specific to …
Figure 3 with 3 supplements
MYPT1 limits kinetochore Plk1 localization and phosphorylation.
(A) U2OS before (CT) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) stained for DNA, centromere-specific human antisera (ACA), tubulin, and Plk1 as indicated. Scale bar, 5 µm. (B) …
Figure 3—figure supplement 1
Whole cell lysates compared for changes in protein expression.
Western blots using whole cell lysates from U2OS before (Control) and after si-RNA-mediated MYPT1 knockdown (MYPT1 KD) for the indicated protein targets. Numbers indicate protein levels relative to …
Figure 3—figure supplement 2
Analysis of Plk1 levels.
U2OS before (CT) and after si-RNA-mediated Cyclin A-knockdown (CycA KD) stained for DNA, centromere-specific human antisera (ACA), and Plk1 as indicated. Scale bar, 5 µm. Quantified intensity …
Figure 3—figure supplement 3
Analysis of pPlk1 levels.
Quantification of the intensity of kinetochore pPlk1-Thr210 staining in U2OS cells before (Control) and after si-RNA-mediated Cyclin A knockdown (Cyc A KD) in prometaphase (Prometa) and metaphase …
Figure 4 with 4 supplements
MYPT1 promotes efficient error correction by regulating Plk1.
(A) Microtubule turnover rates were measured and k-MT half-life was calculated from RPE1 cells before (Control) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) in prometaphase and metaphase …
Figure 4—figure supplement 1
Fluorescence Dissipation After Photoactivation (FDAPA) Representative Curves From Microtubule Stability Measurements.
(Top) Examples of normalized fluorescence dissipation after photoactivation (FDAPA) in prometaphase for untreated (Control) and MYPT1-KD single cells that are representative of the mean. These data …
Figure 4—figure supplement 2
Calcium Stabilization Assay.
(Top) Representative images of assay quantified in (D). Scale bar, 5 µm. (Bottom) Quantification of intensity of spindle tubulin staining in U2OS cells treated with calcium stabilization assay …
Figure 4—figure supplement 3
Quantifications of Other Mechanical Effects of MYPT1 in Mitotic Cells.
Table listing quantification of spindle length and inter-kinetochore distance ±SEM. For spindle length, n ≥ 13 cells per condition as listed; for inter-kinetochore distance, n ≥ 100 kinetochore …
Figure 4—figure supplement 4
Analysis of Whole Cell Levels of Expression of Various MYPT1 Plasmids.
Western blots using whole-cell lysates from U2OS cells before (CT) and after lipofectamine-mediated transfection of either full-length MYPT1, MYPT1-472:473DD, MYPT1-473A, or MYPT1-473D mutant …
Figure 5
Plk1 self-priming of PBIP1 is influenced by Cyclin A/Cdk1 and MYPT1.
(A) Western blots using whole cell lysates from U2OS before (CT) and after si-RNA-mediated Cyclin A-knockdown (CycA KD) of MYPT1 knockdown (MYPT1 KD) for the indicated protein targets. (B) …
Figure 6
MYPT1 cross-regulates Cyclin A/Cdk1-dependent priming and self-priming of Plk1 substrates during mitosis.
Cyclin/Cdk1 has an established role in catalyzing the priming phosphorylation of substrates to promote Plk1 binding as depicted for hypothetical substrate ‘A’. MYPT1 is a specific substrate shown to …
Additional files
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Supplementary file 1
Mitotic phosphoproteome dataset unfiltered and filtered by Cdk1 consensus motif
Complete phosphoproteome dataset, along with dataset filtered by Cdk1 minimum consensus motif. Raw data were searched using COMET in high resolution mode against a target-decoy (reversed) version of the human proteome sequence database, filtered, and quantified, and phosphopeptide ratios were adjusted as described in the Methods section of the text. Significance of log2 phosphopeptide fold-change was determined by two-tailed Student’s t test assuming unequal variance. Dataset was also filtered by the Cdk1 minimum consensus motif (pS/pT-P; (Nigg, 1993; Moreno and Nurse, 1990; Songyang et al., 1994)), listed in the second tab of the file.
- https://doi.org/10.7554/eLife.29303.018
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Supplementary file 2
Phosphoproteome dataset filtered by Aurora B consensus motif
Dataset filtered by the Aurora B kinase consensus motif (R/K – X – pS/pT) (Kim et al., 2010; Meraldi et al., 2004).
- https://doi.org/10.7554/eLife.29303.019
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Supplementary file 3
Phosphoproteome dataset filtered by Plk1 consensus motif
Dataset filtered by the Plk1 kinase consensus motif (D/E-X-pS/pT) (Nakajima et al., 2003; Grosstessner-Hain et al., 2011).
- https://doi.org/10.7554/eLife.29303.020
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Transparent reporting form
- https://doi.org/10.7554/eLife.29303.021
