Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Role of Polo-like kinase 1 in the regulation of the action of p31 comet in the disassembly of mitotic checkpoint complexes
doi: 10.1073/pnas.1902970116
Figure Lengend Snippet: Action of protein kinases on components of the purified p31-TRIP13 system. (A) Effects of protein kinases on the disassembly of Cdc20-Mad2 by the purified p31-TRIP13 system. The recombinant protein kinases were added at the indicated concentrations to a reaction mixture similar to that described for MC disassembly (Materials and Methods), except that extract was replaced by TRIP13 (50 nM) and his6-p31 (0.6 nM). The plot shows values of Mad2 release from MC, relative to incubation without protein kinases. (B) Phosphorylation of components of the p31-TRIP13 system by mitotic protein kinases. The phosphorylation of the following recombinant proteins—GST-p31 (0.4 pmol), TRIP13 (0.4 pmol), and MC (10 pmol)—was determined in a reaction mixture that contained, in a volume of 10 μL: 25 mm Tris⋅HCl (pH 7.2), 15 mM MgCl2, 1 mM DTT, 1 mg/mL BSA, 60 mM β-glycerol phosphate, 15 mM p-nitrophenyl phosphate, and 0.06 mM ATP that contained 2.5 μCi [γ-32P]ATP. The indicated protein kinases were supplemented at the following concentrations (suitable for the phosphorylation of known substrate proteins): 80 nM Plk1; 0.8 nM Bub1-Bub3; and 1.6 nM Cdk1-Cyc B. Following incubation at 37 °C for 30 min, samples were subjected to SDS/PAGE. The incorporation of 32P-phosphate into the relevant proteins was detected by phosphor storage analysis. Electrophoretic migration positions of molecular size marker proteins (kDa) are indicated on the left side.
Article Snippet: We thank Dr. G. Siemeister (Bayer AG) for generously providing a sample of BAY-320 inhibitor of Bub1 and Dr. A. Musacchio for baculovirus expression vector of Bub1-Bub3.
Techniques: Purification, Recombinant, Incubation, Phospho-proteomics, SDS Page, Migration, Marker
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![Action of protein kinases on components of the purified p31-TRIP13 system. (A) Effects of protein kinases on the disassembly of Cdc20-Mad2 by the purified p31-TRIP13 system. The recombinant protein kinases were added at the indicated concentrations to a reaction mixture similar to that described for MC disassembly (Materials and Methods), except that extract was replaced by TRIP13 (50 nM) and his6-p31 (0.6 nM). The plot shows values of Mad2 release from MC, relative to incubation without protein kinases. (B) Phosphorylation of components of the p31-TRIP13 system by mitotic protein kinases. The phosphorylation of the following recombinant proteins—GST-p31 (0.4 pmol), TRIP13 (0.4 pmol), and MC (10 pmol)—was determined in a reaction mixture that contained, in a volume of 10 μL: 25 mm Tris⋅HCl (pH 7.2), 15 mM MgCl2, 1 mM DTT, 1 mg/mL BSA, 60 mM β-glycerol phosphate, 15 mM p-nitrophenyl phosphate, and 0.06 mM ATP that contained 2.5 μCi [γ-32P]ATP. The indicated protein kinases were supplemented at the following concentrations (suitable for the phosphorylation of known substrate proteins): 80 nM Plk1; 0.8 nM <t>Bub1-Bub3;</t> and 1.6 nM Cdk1-Cyc B. Following incubation at 37 °C for 30 min, samples were subjected to SDS/PAGE. The incorporation of 32P-phosphate into the relevant proteins was detected by phosphor storage analysis. Electrophoretic migration positions of molecular size marker proteins (kDa) are indicated on the left side.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5526/pmc06575526/pmc06575526__pnas.1902970116fig02.jpg)