Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins

Background: Signal transduction pathways with shared components must be ins ulated from each other to avoid the inappropriate activation of multiple pa thways by a single stimulus. Scaffold proteins are thought to contribute to this specificity by binding select substrates. Results: We have studied the ability of scaffold proteins to influence sign aling by the yeast kinase Ste11, a MAPKKK molecule that participates in thr ee distinct MAP kinase pathways: mating, filamentation, and HOG. We used pr otein fusions to force Ste11 to associate preferentially with a subset of i ts possible binding partners in vivo, including Ste5, Ste7, and Pbs2. Signa ling became confined to a particular pathway when Ste11 was covalently atta ched to these scaffolds or substrates. This pathway bias was conferred upon both stimulus-activated and constitutively active forms of Ste11. We also used membrane-targeted derivatives of the mating pathway scaffold, Ste5, to show that stimulus-independent signaling initiated by this scaffold remain ed pathway specific. Finally, we demonstrate that loss of pathway insulatio n has a negative physiological consequence, as nonspecific activation of bo th the HOG and mating pathways interfered with proper execution of the mati ng pathway. Conclusions: The signaling properties of these kinase fusions support a mod el in which scaffold proteins dictate substrate choice and promote pathway specificity by presenting preferred substrates in high local concentration. Furthermore, insulation is inherent to scaffold-mediated signaling and doe s not require that signaling be initiated by pathway-specific stimuli or ac tivator proteins, Our results give insight into the mechanisms and physiolo gical importance of pathway insulation and provide a foundation for the des ign of customized signaling proteins.