The order and timing of cell cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDK). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that the cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, the distribution of serines and threonines as phospho-acceptors, and the positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phospho-adaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within the networks of phosphorylation sites in different targets provides a wide range of possibilities for the differential amplification of Cdk1 signals, providing a mechanism to generate a wide range of thresholds in the cell cycle.

The quantitative model of cyclin-dependent kinase (CDK) function states that cyclins temporally order cell cycle events at different CDK activity levels, or thresholds. The model lacks a mechanistic explanation, as it is not understood how different thresholds are encoded into substrates. We show that a multisite phosphorylation code governs the phosphorylation of CDK targets and that phosphorylation clusters act as timing tags that trigger specific events at different CDK thresholds. Using phospho-degradable CDK threshold sensors with rationally encoded phosphorylation patterns, we were able to predictably program thresholds over the entire range of the Saccharomyces cerevisiae cell cycle. We defined three levels of CDK multisite phosphorylation encoding: (i) Ser-Thr swapping in phosphorylation sites, (ii) patterning of phosphorylation sites, and (iii) cyclin-specific docking combined with modulation of CDK activity. Thus, CDK can signal via hundreds of differentially encoded targets at precise times to provide a temporally ordered phosphorylation pattern required for cell division.

Background: Conserved two-component system ColRS of Pseudomonas genus has been implicated in several unrelated phenotypes. For instance, deficiency of P. putida ColRS system results in lowered phenol tolerance, hindrance of transposition of Tn4652 and lysis of a subpopulation of glucose-grown bacteria. In order to discover molecular mechanisms behind these phenotypes, we focused here on identification of downstream components of ColRS signal transduction pathway.

Highlights d Budding yeast G2-cyclin Clb3 uses a PxxPxF motif for specific substrate targeting d PxxPxF promotes Ypr174c phosphorylation to recruit Polo kinase Cdc5 to centrosomes d Hydrophobic patch of cyclins can bind both exclusive and universal docking motifs d Elements around the docking motifs tune their specificity for yeast and human CDKs

Studies on multisite phosphorylation networks of cyclin-dependent kinase (CDK) targets have opened a new level of signaling complexity by revealing signal processing routes encoded into disordered proteins. A model target, the CDK inhibitor Sic1, contains linear phosphorylation motifs, docking sites, and phosphodegrons to empower an N-to-C terminally directed phosphorylation process. Here, we uncover a signal processing mechanism involving multi-step competition between mutually diversional phosphorylation routes within the S-CDK-Sic1 inhibitory complex. Intracomplex phosphorylation plays a direct role in controlling Sic1 degradation, and provides a mechanism to sequentially integrate both the G1-and S-CDK activities while keeping S-CDK inhibited towards other targets. The competing phosphorylation routes prevent premature Sic1 degradation and demonstrate how integration of MAPK from the pheromone pathway allows one to tune the competition of alternative phosphorylation paths. The mutually diversional phosphorylation circuits may be a general way for processing multiple kinase signals to coordinate cellular decisions in eukaryotes.