PHOSPHORYLATION AND LOCALIZATION OF KSS1, A MAP KINASE OF THE SACCHAROMYCES-CEREVISIAE PHEROMONE RESPONSE PATHWAY

Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa hap loids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo. De novo protein synthesis was required for sustained pheromone-i nduced phosphorylation of Kss1. Catalytically inactive Kss1 mutants di splayed alpha-factor-induced phosphorylation on both residues, even in kss1 Delta cells; hence, autophosphorylation is not obligatory for th ese modifications. In kss1 Delta fus3 Delta double mutants, Kss1 phosp horylation was elevated even in the absence of pheromone; thus, cross- phosphorylation by Fus3 is not responsible for Kss1 activation. In con trast, pheromone-induced Kss1 phosphorylation was eliminated in mutant s deficient in two other protein kinases, Ste11 and Ste7. A dominant h yperactive allele of STE11 caused a dramatic increase in the phosphory lation of Kss1, even in the absence of pheromone stimulation, but requ ired Ste7 for this effect, suggesting an order of function: Ste11 --> Ste7 --> Kss1. When overproduced, Kss1 stimulated recovery from pherom one-imposed G(1) arrest. Catalytic activity was essential for Kss1 fun ction in signal transmission, but not for its recovery-promoting activ ity. Kss1 was found almost exclusively in the particulate material and its subcellular fractionation was unaffected by pheromone treatment. Indirect immunofluorescence demonstrated that Kss1 is concentrated in the nucleus and that its distribution is not altered detectably during signaling.