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 determine the sites on the WD-repeat protein Receptor for Activated C Kinase 1 (RACK1) necessary for it to interact with the cAMP-specific phosphodiesterase isoform PDE4D5. Analysis of deletion mutations demonstrated that WD-repeats 5-7, inclusively, of RACK1 contained the ... major site for interaction with 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 blocked the interaction. The ability of these mutations to block the interaction was confirmed by "pull-down" assays using bacterially expressed glutathione-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 acids identified by mutagenesis are clustered in a discrete surface of RACK1. We propose that this surface of RACK1 is the major site for its interaction with the unique amino-terminal region of PDE4D5.
Mesh Terms:
3',5'-Cyclic-AMP Phosphodiesterases, Amino Acid Sequence, Animals, Binding Sites, Cyclic AMP, Cyclic Nucleotide Phosphodiesterases, Type 4, Models, Molecular, Molecular Sequence Data, Peptides, Point Mutation, Repetitive Sequences, Amino Acid, Two-Hybrid System Techniques
3',5'-Cyclic-AMP Phosphodiesterases, Amino Acid Sequence, Animals, Binding Sites, Cyclic AMP, Cyclic Nucleotide Phosphodiesterases, Type 4, Models, Molecular, Molecular Sequence Data, Peptides, Point Mutation, Repetitive Sequences, Amino Acid, Two-Hybrid System Techniques
Cell. Signal.
Date: Jul. 01, 2001
PubMed ID: 11516626
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