Fig. 3: Phospho-pocket mutations reduce phosphorylation of APC/C subunits in vitro.
From: Phosphate-binding pocket on cyclin B governs CDK substrate phosphorylation and mitotic timing
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 studies37. 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. 4 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. 6. Source data are provided as a Source Data file.
