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 identifie d 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 stimulati on of the cells with the phorbol ester phorbol S-myristate S-acetate. PDE4D 5 did not interact with two other WD-repeat proteins, beta'-coatomer protei n and G(s)beta, in two-hybrid tests. RACK1 did not interact with other PDE4 D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms, PDE4D5 and RACK1 interacted with high affinity (K-a approximately 7 pM) when they were expr essed and purified from Escherichia coli, demonstrating that the interactio n does not require intermediate proteins, The binding of the E. coli-expres sed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but ca used a 3-4-fold change in its sensitivity to inhibition by the PDE4 selecti ve inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 we re extremely similar, with the major amount of both proteins (70%) in the h igh speed supernatant (S2) fraction. Analysis of constructs with specific d eletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino terminal region of PDE4D5 was n ecessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling com plex.