Cdc28 and Fus3 synergistically regulate mating pheromone signaling dynamics via a shared multi-site phosphorylation region on the scaffold protein Ste5

COLMAN-LERNER, ALEJANDRO; BUSH, ALAN; AMMERER, GUSTAV; WINTERS, MATTHEW J.; HOLLENSTEIN, DAVID MARIA; REPETTO, MARÍA VICTORIA; REITER, WOLFGANG; PRYCIAK, PETER M.

Stanford, CA

Encuentro; Yeasts Genetics Meeting; 2018

Genetics Society of America

Normal cell behavior requires the ability to detect and respond to extracellular signals. To study how cells integrate thesesignals with the internal cell state, we used as a model system the response to mating pheromone in the budding yeast S.cerevisiae, which has an antagonistic relationship with the cell cycle. The pheromone response pathway MAPK (Fus3) arreststhe cell cycle by activating the CDK inhibitor Far1. Conversely, when cells pass START, the CDK (Cln2-Cdc28) blocks pheromoneresponse by phosphorylating the scaffold protein Ste5 near a plasma membrane binding (PM) domain, which prevents itsassociation with the plasma membrane. Here, using quantitative time-lapse microscopy, we examined Ste5 membranerecruitment dynamics at different cell cycle stages. Surprisingly, in S-phase, where Ste5 recruitment should be blocked,pheromone stimulation caused an initial recruitment followed by a steep drop-off. This delayed inhibition revealed arequirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry,and electrophoretic analyses suggested that the CDK and MAPK modify shared sites flanking the PM domain, and that thesesites are most extensively phosphorylated when both kinases are active and able to bind their docking sites on Ste5. Ourresults suggest that the synergy between these kinases depends on a priming role of Fus3, in which it phosphorylatesthreonine residues. Then, these pThr residues become docking sites for the Cks1 subunit of CDK, which then proceeds tocomplete the phosphorylation of the remaining sites. Functionally, one consequence of the joint action by two kinases (asopposed to solo control by CDK alone) is to broaden the time-window in the cell cycle during which the CDK can effectively 36block the mating response. Another is to make this inhibition more sensitive to other inputs or cell states that can regulateFus3 in a way that reduces or enhances CDK control of mating. In conclusion, our findings indicate that the Ste5 scaffoldprotein behaves as a regulatory node whereby distinct kinases can target a shared set of phosphorylation sites to jointlycontrol the affinity of a membrane interaction domain. The net impact on the signaling pathway can range from a mildmodulation to severe quenching, depending on the regulatory inputs. These properties highlight how multiplephosphorylation sites, with different probabilities of being phosphorylated, plus cooperative interactions between kinases,can expand the options for kinase-mediated regulation.