THE MAP KINASE FUS3 ASSOCIATES WITH AND PHOSPHORYLATES THE UPSTREAM SIGNALING COMPONENT STE5

Activation of the Saccharomyces cerevisiae MAP kinase Fus3 is thought to occur via a linear pathway involving the sequential action of three proteins: Ste5, a protein of unknown function, Ste11, a MAPKK kinase homolog, and Ste7, a MAPK kinase homolog which phosphorylates and acti vates Fus3. In this report, we present evidence for a novel mechanism of Fus3 activation that involves a direct association with Ste5, a pro tein not predicted to interact with Fus3. First, overexpression of Ste 5 suppresses fus3 point mutations in an allele-specific manner and inc reases Fus3 kinase activity in vitro. Second, Ste5 associates with Fus 3 in vivo as demonstrated by the two-hybrid system and by two methods of copurification. Third, Ste5 and Fus3 associate prior to pheromone s timulation even when Fus3 is inactive, and in strains lacking Ste7 and Ste11. Fourth, Ste5 is phosphorylated by Fus3 in purified complexes a nd copurifies with an additional protein kinase(s). These observations suggest the possibility that Ste5 promotes signal transduction by tet hering Fus3 to its activating protein kinase(s).