Journal: Nature Communications

Article Title: Phosphate-binding pocket on cyclin B governs CDK substrate phosphorylation and mitotic timing

doi: 10.1038/s41467-025-59700-7

Figure Lengend Snippet: a APC/C was immunoprecipitated from yeast lysate and treated with wild-type or clb2-pp Clb2-Cdk1 plus mutant Cks1 and radiolabeled ATP. Reaction products were analyzed by SDS-PAGE and autoradiography (lane 1: kinase only control; lane 2: no kinase control; lane 3-6: wild-type Clb2; lane 7-10: Clb2-pp). APC/C subunits were identified based on previous studies . b 2.5 μM purified Cdc16 fragment (aa 31–180) was incubated with 150 nM wild-type or clb2-pp Clb2-Cdk1 plus wild-type or mutant Cks1 and radiolabeled ATP. Diagrams at the top indicate suboptimal (S/T*-P; yellow) CDK consensus sites (S: circle; T: triangle) in the fragment (see Supplementary Fig. for complete sequences). Representative of 10 independent experiments. c Same as ( b ) with 2.5 μM purified Cdc27 fragment (aa 241–360) and 100 nM Clb2-Cdk1. Representative of 8 independent experiments. Coomassie Blue-stained gels for ( b and c ) are found in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: 50–200 nM Clb2-Cdk1, equimolar Cks1, 50 nCi [γ− 32 P]-ATP (Hartmann Analytic), and 2.5–10 μM substrate were incubated in kinase buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 100 μM ATP) at 25 °C.

Techniques: Immunoprecipitation, Mutagenesis, SDS Page, Autoradiography, Control, Purification, Incubation, Staining

Journal: Nature Communications

Article Title: Phosphate-binding pocket on cyclin B governs CDK substrate phosphorylation and mitotic timing

doi: 10.1038/s41467-025-59700-7

Figure Lengend Snippet: a , b , 2.5 μM of the indicated Cdc27 mutant fragments were incubated with 100 nM wild-type or clb2-pp Clb2-Cdk1 plus mutant Cks1 and radiolabeled ATP. Reaction products were analyzed by Phos-tag SDS-PAGE and autoradiography. Diagrams at the top indicate suboptimal (S/T*-P; yellow) CDK consensus sites (S: circle; T: triangle) in the tested fragment (see Supplementary Fig. for complete sequences). Representative of 4 independent experiments. Coomassie Blue-stained gels in Supplementary Fig. . For the graph at the bottom of panel ( a ), phosphate incorporation into the single-site mutants over time was used to calculate the mean phosphorylation rate (± SD) in 4 independent experiments. Source data are provided as a Source Data file.

Article Snippet: 50–200 nM Clb2-Cdk1, equimolar Cks1, 50 nCi [γ− 32 P]-ATP (Hartmann Analytic), and 2.5–10 μM substrate were incubated in kinase buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 100 μM ATP) at 25 °C.

Techniques: Mutagenesis, Incubation, SDS Page, Autoradiography, Staining, Phospho-proteomics

Journal: Nature Communications

Article Title: Phosphate-binding pocket on cyclin B governs CDK substrate phosphorylation and mitotic timing

doi: 10.1038/s41467-025-59700-7

Figure Lengend Snippet: 2.5 μM of purified Bud6 fragment (aa 126–360) or 5 μM of purified Spa2 fragment (aa 524–670) was incubated with 100 nM wild-type or clb2-pp Clb2-Cdk1 plus wild-type or mutant Cks1 and radiolabeled ATP. Diagrams at top indicate optimal (S/T*-P-x-K/R; green) and suboptimal (S/T*-P; yellow) CDK consensus sites (S: circle; T: triangle) in the tested fragment (see Supplementary Fig. for complete sequences). Representative of 3 independent experiments. Coomassie Blue-stained gels in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: 50–200 nM Clb2-Cdk1, equimolar Cks1, 50 nCi [γ− 32 P]-ATP (Hartmann Analytic), and 2.5–10 μM substrate were incubated in kinase buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 100 μM ATP) at 25 °C.

Techniques: Purification, Incubation, Mutagenesis, Staining

Journal: Nature Communications

Article Title: Phosphate-binding pocket on cyclin B governs CDK substrate phosphorylation and mitotic timing

doi: 10.1038/s41467-025-59700-7

Figure Lengend Snippet: The indicated five substrates (10 μM) were incubated for 60 min in 55 μl reaction volumes containing 200 nM wild-type or clb2-pp Clb2-Cdk1 plus 200 nM mutant Cks1. Samples were trypsinized, and seven aliquots from each reaction were subjected to LC-MS/MS. High-confidence phosphopeptides (2% ion intensity, AScore 15) were tabulated with the PTM Profiles function in PEAKS software, which provides the chromatographic peak areas for modified peptides. To normalize for the quantity of substrate, modified peptide peak areas were divided by the total peak area of peptides from the Smt3 tag at the amino-terminus of the substrate. Normalized quantities are plotted here (mean +/− SD; n = 4–7 replicates; see Supplementary Data ). Asterisks indicate results of two-tailed unpaired t-tests (* p < 0.05; ** p < 0.01). Diagrams at the top indicate optimal (S/T*-P-x-K/R; green) and suboptimal (S/T*-P; yellow) CDK consensus sites. Blue indicates non-consensus sites lacking a proline at + 1 but containing a basic (K/R) residue at + 3 or + 4; other non-consensus sites are colored gray (see Supplementary Fig. for complete sequences). A small number of CDK consensus sites were not confidently quantified and are shown as vertical marks (S95 in Cdc16, S283 in Bud6, S544 and T549 in Spa2). Source data are provided in Supplementary Data and the Source Data file.

Article Snippet: 50–200 nM Clb2-Cdk1, equimolar Cks1, 50 nCi [γ− 32 P]-ATP (Hartmann Analytic), and 2.5–10 μM substrate were incubated in kinase buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 100 μM ATP) at 25 °C.

Techniques: Incubation, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Software, Modification, Two Tailed Test, Residue