Positively charged specificity site in cyclin B1 is essential for mitotic fidelity

Phosphorylation of substrates by cyclin-dependent kinases (CDKs) is the driving force of cell cycle progression. Several CDK-activating cyclins are involved, yet how they contribute to substrate specificity is still poorly understood. Here, we discovered that a positively charged pocket in cyclin B1, which is exclusively conserved within B-type cyclins and binds phosphorylated serine- or threonine-residues as well as acidic side chains, is essential for correct execution of mitosis. HeLa cells expressing pocket mutant cyclin B1 are strongly delayed in anaphase onset due to multiple defects in mitotic spindle function and timely activation of the E3-ligase APC/C. Non-functional pocket results in decreased APC/C phosphorylation at non-consensus CDK1 sites, compromised in vitro ubiquitylation activity, and reduced binding of the E2-enzyme UBE2S. Our results support a model in which cyclin B1’s pocket serves as specificity factor for sequential substrate phosphorylations involving initial priming events facilitating subsequent pocket-dependent phosphorylations at non-consensus CDK1 motifs.