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.
Steele
M R
MR
Veterans Affairs Medical Center, Huntsman Cancer Institute, Department of Medicine (Division of Oncology) and Oncological Science, University of Utah Health Sciences Center, 500 Foothill Drive, Salt Lake City, UT 84148, USA.
McCahill
A
A
Thompson
D S
DS
MacKenzie
C
C
Isaacs
N W
NW
Houslay
M D
MD
Bolger
G B
GB
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D000075370:Receptors for Activated C Kinase; D020449:Repetitive Sequences, Amino Acid; D020798:Two-Hybrid System Techniques
D010455:Peptides; D000075370:Receptors for Activated C Kinase; C070301:peptide I; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4