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

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.

Single-cell profiling screen identifies microtubule-dependent reduction of variability in signaling.

Populations of isogenic cells often respond coherently to signals, despite differences in protein abundance and cell state. Previously, we uncovered processes in the Saccharomyces cerevisiae pheromone response system (PRS) that reduced cell-to-cell variability in signal strength and cellular response. Here, we screened 1,141 non-essential genes to identify 50 "variability genes". Most had distinct, separable effects on strength and variability of the PRS, defining these quantities as genetically distinct "axes" of system behavior. Three genes affected cytoplasmic microtubule function: BIM1, GIM2, and GIM4 We used genetic and chemical perturbations to show that, without microtubules, PRS output is reduced but variability is unaffected, while, when microtubules are present but their function is perturbed, output is sometimes lowered, but its variability is always high. The increased variability caused by microtubule perturbations required the PRS MAP kinase Fus3 and a process at or upstream of Ste5, the membrane-localized scaffold to which Fus3 must bind to be activated. Visualization of Ste5 localization dynamics demonstrated that perturbing microtubules destabilized Ste5 at the membrane signaling site. The fact that such microtubule perturbations cause aberrant fate and polarity decisions in mammals suggests that microtubule-dependent signal stabilization might also operate throughout metazoans.