Identification of a surface on the beta-propeller protein RACK1 that interacts with the cAMP-specific phosphodiesterase PDE4D5

A strategy of mutagenesis followed by yeast two-hybrid assay was used to de termine the sites on the WD-repeat protein Receptor for Activated C Kinase 1 (RACK1) necessary for it to interact with the cAMP-specific phosphodieste rase isoform PDE4D5. Analysis of deletion mutations demonstrated that WD-re peats 5-7, inclusively, of RACK1 contained the major site for interaction w ith PDE4D5. A reverse two-hybrid screen focusing on WD-repeats 5-7 of RACK1 isolated 11 single amino acid mutations from within this region that block ed the interaction. The ability of these mutations to block the interaction was confirmed by "pull-down" assays using bacterially expressed glutathion e-S-transferase (GST)-RACK1 and mammalian cell-expressed PDE4D5. A model of RACK1 structure. based on the structural similarity of RACK1 to other beta -propeller WD-repeat proteins. indicated that the majority of the amino ac ids identified by mutagenesis are clustered in a discrete surface of RACK1. We propose that this surface of RACK1 is the major site for its interactio n with the unique aminoterminal region of PDE4D5. (C) 2001 Elsevier Science Inc. All rights reserved.