A processive phosphorylation circuit with multiple kinase inputs and mutually diversional routes controls G1/S decision

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.


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Policy information about availability of computer code Data collection Live-cell microscopy data was collected using ZEN software (Zeiss, version 2.3, blue edition). The 32-P autoradiography images from kinase assays were quantified by using ImageQuant TL software (Amersham, version 8.1), as described in methods section.

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Microscopy data was analyzed by using custom made scripts for image segmentation, cell tracking and quantification MATLAB (Mathworks, version R2016a). The scripts are available upon request.
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No sample-size pre-calculations were made. For kinase assays, all experiments were repeated independently at least twice due to very low variability between the experiments. The low (<10%) variability was further corroborated in reactions with WT-Sic1 where the number of independent experiments performed was in some cases as high as 6. Therefore, and due to highly defined biochemical composition and defined reaction conditions, we were sufficiently satisfied with two independent experiments. In addition, all independent kinase assays were processed in two or more timepoints, which were not regarded as independent replicas, but further demonstrated very low technical variability of the assay. For in vivo experiments where the readout was processed in form of immunological detection (Western blot), all experiments were carried out at least twice, including preparing and processing cell cultures, applying experimental conditions for the cultures, recovering samples and processing them until the final signal detection and quantification.
In live-cell microscopy experiments with unperturbed cell cycle 50 cells from two distinct individual colonies were considered to be sufficient for the analysis, as the median values and their 95% confidence intervals showed very little variation (<10%) between different individual colonies of the same strain, and combined 95% confidence interval values were reasonably narrow. For mating pheromone intercepted livecell microscopy experiments 15 individual cells from at least 5 distinct experiments with independent colonies were considered to be sufficient for the analysis, if obtained 95% confidence interval values were reasonably narrow. In some cases more cells were analyzed if more experiemntal data was available.
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All data presented is from at least two replicate experiments, all replication events were successful and are included in the data.
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Validation
The anti-HA antibody from Covance was raised against the twelve amino acid peptide CYPYDVPDYASL in mous and veryfied by Wester blotting against Hemagglutinin tag. The anti-Cks1 antibody from Labas AS was raised against the full Cks1 protein in rabbit and verified by western blotting against the bacterially expressed and purified Cks1. The anti-Cdk1 antibody (Cdc28 Antibody, yC-20) from SantaCruz Biotechnology was raised against the C-terminal region of yeast Cdk1 protein in goat and verified by western blotting against the yeast Cdk1 protein.