The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform.
The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen. The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines. The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate. PDE4D5 did not interact with two other WD-repeat proteins, beta'-coatomer protein and Gsbeta, in two-hybrid tests. RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms. PDE4D5 and RACK1 interacted with high affinity (Ka approximately 7 nM) [corrected] when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins. The binding of the E. coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3-4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction. Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D007527:Isoenzymes; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D011233:Precipitin Tests; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity; D015003:Yeasts / Q000201:enzymology