Cyclin A and Cks1 promote kinase consensus switching to non-proline directed CDK1 phosphorylation

Ordered protein phosphorylation by CDKs is a key mechanism for regulating the cell cycle. How temporal order is enforced in mammalian cells remains unclear. Using a fixed cell kinase assay and phosphoproteomics, we studied how CDK1 activity and non-catalytic CDK1 subunits contribute to the choice of substrate and phosphorylation site. Increases in CDK1 activity alters substrate choice, with intermediate and low sensitivity CDK1 substrates enriched in DNA replication and mitotic functions, respectively. This activity dependence was shared between Cyclin A- and Cyclin B-CDK1. Cks1 has a proteome-wide role as an enhancer of multisite CDK1 phosphorylation. Contrary to the model of CDK1 as an exclusively proline-directed kinase, we show that Cyclin A and Cks1 promote non-proline directed phosphorylation, preferably on sites with a +3 lysine residue. Indeed, 70% of cell cycle regulated phosphorylations, where the kinase carrying out this modification has not been identified, are non-proline directed CDK1 sites.