505.13 - Quantitative Mass Spectrometry Reveals a Proteome-wide Role for Cyclin A and Cks1 in Multisite, Non-Proline Directed Phosphorylation by CDK1

Tony Ly (University of Dundee), Aymen Al-Rawi (University of Dundee), Jane Endicott (University of Newcastle), Svitlana Korolchuk (University of Newcastle)

Cdk1 is a master kinase of mitotic cell division, phosphorylating thousands of substrates in a highly temporally regulated manner. How Cdk1 phosphorylates the right substrate at the right time remains an open question.

Cdk1 activity requires complex formation with a cyclin subunit: cyclin A or cyclin B. Additionally, active Cdk1 is found in complex with a small adaptor protein called Cks1. Cyclin subunits activate Cdk1 and tune Cdk1 substrate choice through interactions with short linear motifs on substrates. While Cks1 is essential in all organisms tested to date, a general function for Cks1 in Cdk1 substrate choice has not been demonstrated.

In this study, we investigated the roles of the non-catalytic subunits of Cdk1 in substrate phosphorylation in human cells. We designed an in vitro assay in which we induced protein phosphorylation in fixed and permeabilized human lymphoblasts using active recombinant Cdk1 either in complex with cyclin A, cyclin B, or cyclin B+Cks1. Global phosphorylation levels were then measured using quantitative mass spectrometry.

Our data demonstrate that, contrary to the textbook model of Cdk1 as a proline-directed kinase, both cyclin A and Cks1 facilitate widespread Cdk1 phosphorylation of sites lacking a +1 proline motif. Our results also suggest a role for Cks1 in multisite phosphorylation. We propose a model whereby cyclin A and Cks1 have indispensable functions in promoting non-proline directed, multisite phosphorylation of Cdk1 substrates to mediate dynamic, switch-like changes in protein function required for mitotic entry, proper chromosome segregation and cell division.

This work was supported by a Wellcome Trust and Royal Society Sir Henry Dale Fellowship To T.L. (206211/A/17/Z).