Fig. 4: Cyclin B1’s phosphate-binding pocket is important for APC/C phosphorylation at non-consensus CDK1 sites.

A In vitro kinase assay using recombinant CCC^wt/mut to phosphorylate recombinant strep-tagged apo-APC/C and MBP-Emi2^NT, both present in the same kinase reaction mixture. Shown are western blots for all relevant proteins involved in the reaction. As readout for phosphorylation efficiency, pT97 (MBP-Emi2^NT) and APC3 (apoAPC/C) phosphorylation pattern were used. APC3 and APC1 blots were conducted on PhosTag™ gels. In vitro kinase reactions were performed at 30 °C, and at indicated timepoints samples were taken and analyzed by WB. B Pie charts and discrete heatmaps of APC/C phosphosites identified after in vitro phosphorylation by CCC^wt/mut. Dephosphorylated and purified APC/C has been subjected to an in vitro phosphorylation assay using either cyclin B1^wt or cyclin B1^mut and phosphosites have been identified by MS. The phosphosites were stringently filtered (see Fig. S4 for details). Then, the obtained phosphosites from CCC^wt were divided in either minimal CDK1 (S/T)P consensus sites (blue) or non-consensus sites (orange). The upper pie chart shows the percentage and number of identified consensus or other phosphosites in the CCC^wt samples. Using these phosphosites as basis, the CCC^mut data set was analyzed and classified in sites that were equally well detected in CCC^mut samples (left) and sites that were either rarely (at least 2 fold reduced in intensity and in half or less than half of the raw files compared to wt; middle) or not at all detected (right). Below the pie charts heatmaps of the identified phosphosites are shown (red: present; light red: reduced at least 2 fold and in half or less than half of the rawfiles compared to CCC^wt; gray: absent. C Bar plot of the fold changes of APC/C subunits, CDC20, cyclin B1 and UBE2S of the immunopurified APC/C of cells overexpressing either cyclin B1^wt or B1^mut. APC3 was immunopurified from siB1&B2 cells expressing either cyclin B1^wt or B1^mut and interaction partners were quantified by MS. For proteins of interest, the ratio of the quantified protein intensities between cyclin B1^wt and cyclin B1^mut was calculated (log₂ intensities cyclin B1^wt – log₂ intensities B1^mut) and normalized on the ratio of the bait protein APC3. The x-axis shows the normalized log₂ changes of relevant indicated proteins (y-axis). The experiment was conducted in n = 3 independent biological replicates, each dot represents a single measurement. Data are represented as mean values ±SD. D Model of pocket-dependent multisite substrate phosphorylations. Step 1: Priming phosphorylation carried out by any kinase creates a docking site for cyclin B1’s phosphate-binding pocket. Step 2: Upon pocket-dependent recruitment, CCC catalyzes the phosphorylation of an additional site. Step 3: This phosphosite can serve as docking site for CCC itself using cyclin B1 or CKS1 as phosphate adaptor or other kinases with phosphate adaptor properties such as casein kinase 1 (CK1) or PLK1. For illustration, CCC (PDB: 7NJ0) is shown in blue (CDK1), orange (CKS1), and green (cyclin B1), PLK1 (AlphaFold2 prediction) in red, and CK1 (PDB: 6GZD) in purple.