Fig. 3: The phosphorylation site-specificity is lost within the inhibitory complex.

a The stoichiometry of intracomplex phosphorylation of Sic1 in vitro was determined by using inhibitory tyrosine kinase Swe1. Mono-phosphorylation of Y19-Cdk1 by Swe1 was used as a quantitative reference to estimate the number of phosphates added per molecule of Sic1 in a separate S-CDK (Clb5–Cdk1–Cks1) kinase assay loaded on the same gel. Bars represent the mean level derived from the data of two values that are obtained from two independent experiments. The values are plotted individually by the open circles. b The stoichiometry for intracomplex phosphorylation of Sic1 in the presence or absence of Cks1 in the assay mixture. The dotted lines represent standard deviation of the time-course obtained from two independent experiments. c Plots demonstrating the Clb5–Cdk1–Cks1–Sic1 intracomplex phosphorylation dynamics of Sic1 versions containing only selected CDK sites with the rest of the CDK sites mutated to alanines. The plots obtained from two independent experiments were nearly overlapping. d The individual phosphorylation profiles shown on c plotted together with the wild-type Sic1 phosphorylation profile for comparison. The 9AP denotes the version with all CDK sites mutated to alanines. The dotted lines represent standard deviation of the time-course obtained from two independent experiments. The full Sic1 amino acid sequences can be found in Source data file. e In vitro phosphorylation of Sic1ΔC versions containing the indicated CDK sites with the rest of the CDK sites mutated to alanines to demonstrate the Cks1-mediated double-phosphorylation of diversionary sites using Clb5–Cdk1–Cks1 complex. Autoradiographs of Phos-tag SDS–PAGE are shown. f Plot demonstrating the dephosphorylation of Sic1 within Sic1–Clb5–Cdk1–Cks1 complex by lambda phosphatase. The plotted trendlines were obtained by fitting the data points as described in the Methods section. The plots obtained from two independent experiments were overlapping. The experiments for a–f were performed twice with similar result. The full gel radiographs are presented in Supplementary Fig. 7. Source data are provided as a Source Data file.