PROTEIN-PROTEIN INTERACTIONS IN THE YEAST PKC1 PATHWAY - PKC1P INTERACTS WITH A COMPONENT OF THE MAP KINASE CASCADE

The two-hybrid system for the identification of protein-protein intera ctions was used to screen for proteins that interact in vivo with the Saccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in the PKC1-me diated signalling pathway. Subsequently, Pkc1p and the other PKC1 path way components encoding members of a MAP kinase cascade, Bck1p (a MEKK ), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP k inase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p , and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, how ever, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction requi red only the predicted C-terminal catalytic domain of Mkk1p. Furthermo re, we detected protein-protein interactions between two Bck1p molecul es via their N-terminal regions. Finally, Mkk2p and Mpk1p also interac ted in the two-hybrid assay. These results suggest that the members of the PKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component o f this pathway.