SIGNALING IN THE YEAST PHEROMONE RESPONSE PATHWAY - SPECIFIC AND HIGH-AFFINITY INTERACTION OF THE MITOGEN-ACTIVATED PROTEIN (MAP) KINASES KSS1 AND FUS3 WITH THE UPSTREAM MAP KINASE KINASE STE7

Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), a nd Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone re sponse pathway of Saccharomyces cerevisiae. To investigate the potenti al role of specific interactions between these enzymes during signalin g, their ability to associate with each other was examined both in sol ution and in vivo. When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (K-d of similar to 5 nM) with Ste 7 in the absence of any additional yeast proteins. These complexes wer e specific because neither Hog1 nor Mpk1 (two other yeast MAPKs), nor mammalian Erk2, was able to associate detectably with Ste7. Neither th e kinase catalytic core of Ste7 nor the phosphoacceptor regions of Ste 7 and Kss1 were necessary for complex formation. Ste7-Kss1 (and Ste7-F us3) complexes were present in yeast cell extracts and were undiminish ed in extracts prepared from a ste5 Delta ste11 Delta double mutant st rain. In Ste7-Kss1 (or Ste7-Fus3) complexes isolated from naive or phe romone-treated cells, Ste7 phosphorylated Kss1 (or Fus3), and Kss1 (or Fus3) phosphorylated Ste7, in a pheromone-stimulated manner; dissocia tion of the high-affinity complex was shown to be required for either phosphorylation event. Deletions of Ste7 in the region required for it s stable association with Kss1 and Fus3 in vitro significantly decreas ed (but did not eliminate) signaling in vivo. These findings suggest t hat the high-affinity and active site-independent binding observed in vitro facilitates signal transduction in vivo and suggest further that MEK-MAPK interactions may utilize a double-selection mechanism to ens ure fidelity in signal transmission and to insulate one signaling path way from another.