Kss1 and Fus3 are MAP kinases (MAPKs or ERKs) in the pheromone response pathway of S. cerevisiae. Stable protein-protein interactions involving these kinases contribute to several aspects of signaling. Kss1 and Fus3 each bind to Ste7, their activating MAPK/ERK kinase (MEK), in the absence of any additional yeast proteins. Other yeast MAP kinases do not bind to Ste7. The Ste7-MAPK complex displays a higher affinity (Kd = 5nM) and stability (half-life = 2 min) than would be expected from a prototypical enzyme-substrate interaction. Complex formation requires a conserved motif in the N-terminus of Ste7, does not require active site-phosphoacceptor residue contacts, and apparently does not position the proteins in an enzyme-substrate orientation. The high-affinity and active site-independent MEK-MAPK complex potentiates signaling in vivo, and provides a "double-selection" to insulate one signaling pathway from another. In addition to binding to their upstream activator, Kss1 and Fus3, whether activated or unactivated, also bind to several of their downstream substrates. Because of this, MAPK activation exhibits switch-like behavior. Kss1 and Fus3 exhibit distinct substrate preferences in vitro which correlate with their different physiological properties in vivo. The network of protein-protein interactions involving MAP kinases contributes to signal transmission, amplification, dissemination and insulation.