A POSITIVE GENETIC SELECTION FOR DISRUPTING PROTEIN-PROTEIN INTERACTIONS - IDENTIFICATION OF CREB MUTATIONS THAT PREVENT ASSOCIATION WITH THE COACTIVATOR CBP

The Escherichia coli let-repressor (TetR) operator system was used to develop a variation of the yeast two-hybrid assay in which disruptions of protein-protein interactions can be identified by a positive selec tion, This assay, designated the ''split-hybrid system,'' contains a t wo-component reporter, The first component contains LexA binding sites upstream of the TetR gene and the second contains TetR operator bindi ng sites upstream of HIS3, Interaction of one protein fused to the Les A DNA binding domain with a second protein fused to the VP16 activatio n domain results in TetR expression. TetR subsequently binds to the le t operators, blocking the expression of HIS3 and preventing yeast grow th in media lacking histidine, The utility of the split-hybrid system was analyzed by examining the phosphorylation dependent interaction of CREB and its coactivator CREB binding protein (CBP). CREB and CBP ass ociate through an interaction that depends upon CREB phosphorylation a t Ser-133, Mutation of this phosphorylation site prevents yeast growth in the standard two-hybrid assay but allows growth in the split-hybri d strains, The split-hybrid system was used to identify other CREB mut ations that disrupt its association with CBP, These mutations localize d around the site of CREB phosphorylation, indicating that only a smal l portion of the CREB activation domain is required for CBP interactio n, The yeast split-hybrid system should be useful in identifying mutat ions, proteins, peptides, and drugs that disrupt protein-protein inter actions.