CDK and MAPK Synergistically Regulate Signaling Dynamics via a Shared Multi-site Phosphorylation Region on the Scaffold Protein Ste5

• Published in: Molecular cell 2018-03, Vol.69 (6), p.938-952.e6
• Main Authors: Repetto, María Victoria, Winters, Matthew J., Bush, Alan, Reiter, Wolfgang, Hollenstein, David Maria, Ammerer, Gustav, Pryciak, Peter M., Colman-Lerner, Alejandro
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Binding Sites; Cdc28; Cell Cycle Checkpoints; Cell Membrane - enzymology; Cks1; Cln2; cyclin; Cyclin-Dependent Kinases - genetics; Cyclin-Dependent Kinases - metabolism; Cyclins - genetics; Cyclins - metabolism; G protein; Kinetics; mating; Mitogen-Activated Protein Kinases - genetics; Mitogen-Activated Protein Kinases - metabolism; pheromone; Phosphorylation; Protein Binding; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Signal Transduction; start; Ste4; Substrate Specificity
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2018.02.018

We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein Ste5 controls signaling through a G1 arrest pathway. Upon cell-cycle entry, CDK inhibits Ste5 via multiple phosphorylation sites, disrupting its membrane association. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell-cycle stages. Surprisingly, in S phase, where Ste5 recruitment should be blocked, we observed an initial recruitment followed by a steep drop-off. This delayed inhibition revealed a requirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry, and electrophoretic analyses suggest that the CDK and MAPK modify shared sites, which are most extensively phosphorylated when both kinases are active and able to bind their docking sites on Ste5. Such collaborative phosphorylation can broaden regulatory inputs and diversify output dynamics of signaling pathways. [Display omitted] •Single-cell assays of Ste5 recruitment reveal cell-cycle-stage-specific dynamics•A CDK and a MAPK jointly regulate Ste5 recruitment by targeting the same phosphosites•Cell-cycle regulation of Ste5 membrane localization requires MAPK negative feedback•Joint kinase control can modulate function over a range of quantitative degrees CDKs and MAPKs phosphorylate similar sites yet generally have distinct functions and substrates. Repetto et al. uncover a case where these kinases collaborate to regulate a substrate in a signal transduction pathway by phosphorylating a shared set of sites.