The RACK1 Signaling Scaffold Protein Selectively Interacts with the cAMP-specific Phosphodiesterase PDE4D5 Isoform

Abstract

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, ␤'-coatomer protein and G s ␤, 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 (K a approximately 7 pM) 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.Modulation of the levels of the second messenger cAMP in cells is important in the regulation of numerous physiological processes, including those in the immune/inflammatory systems, vascular smooth muscle, and the brain. Cyclic nucleotide phosphodiesterases (PDEs) 1 are a diverse family of enzymes that hydrolyze cAMP and cGMP and thus play an important role in modulating cAMP levels (1). The cAMP-specific phosphodiesterases (PDE4s) can be differentiated from other PDEs by sequence homology of the catalytic region of the enzymes (2) and by their ability to be specifically inhibited by the drug rolipram. Rolipram and other specific PDE4 inhibitors have been shown to have anti-depressant, anti-inflammatory, and smooth muscle relaxant activity in humans (2). The PDE4 enzymes are also characterized by the presence of unique regions of amino acid sequence outside the catalytic region of the proteins, which are called upstream conserved regions 1 and 2 (UCR1 and UCR2) and are located in the amino-terminal half of the proteins (3). The PDE4s are comprised of a large family of isoforms, encoded by four different genes (PDE4A, PDE4B, PDE4C, and PDE4D) in humans, with additional diversity being generated by alternative mRNA splicing (2). We and other groups (3-5) have characterized five different isoforms encoded by the human PDE4D gene, all of which appear to be conserved among mammals (6, 7). The five isoforms differ by the substitution of unique blocks of amino acids at the...

Adaptor proteins in protein kinase C-mediated signal transduction

“…The interaction was con®rmed by co-immunoprecipitation of PDE4D5 with native RACK1 in Cos7 cells. No binding is observed with other WD40 proteins including eRACK (Yarwood et al, 1999). Although, interaction of PDE4D5 with RACK1 is not a ected by PMAstimulation of the cells, binding of PDE4D5 to RACK1 increases the sensitivity of the enzyme to its inhibitor, rolipran (Yarwood et al, 1999).…”

“…A yeast two-hybrid screen also demonstrated that RACK1 binds the cAMP phosphodiesterase isoform 4D5 (PDE4D5) (Yarwood et al, 1999). The interaction was con®rmed by co-immunoprecipitation of PDE4D5 with native RACK1 in Cos7 cells.…”

Epigenetic Regulation of BDNF Expression via the Scaffolding Protein RACK1

“…The scaffolding protein RACK1, which was originally identified as an anchoring protein of protein kinase C ␤II (2), belongs to the WD40 family of proteins characterized by seven WD40 repeats forming a seven-blade ␤-propeller structure (3,4). This particular structure allows RACK1 to interact with various enzymes including Fyn kinase (5), focal adhesion kinase (6), receptor protein tyrosine phosphatase (7), the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 (8), as well as the intracellular tails of receptors such as the insulin-like growth factor 1 receptor (IGF-1R) (9,10), the inositol 1,4,5-triphosphate receptor (11), and ion channels such as the NR2B subunit of the N-methyl D-aspartate receptors (5). These interactions, and others, put RACK1 at a focal point for spatial and temporal regulation of various signaling cascades.…”

Function of p73, not of p53, is inhibited by the physical interaction with RACK1 and its inhibitory effect is counteracted by pRB

“…The physical interaction between RACK1 and integrin b subunit or Src is dependent on the stimulation of cells with phorbol ester PMA (Liliental and Chang, 1998;Chang et al, 2001), suggesting that PKC activation is required for the above interaction. On the other hand, the interaction of RACK1 with the common b-chain of the cytokine receptors, cAMPspecific phosphodiesterase isoform PDE4D5 or BZLF1 is constitutive and unaffected in the presence of PMA (Geijsen et al, 1999;Yarwood et al, 1999;Baumann et al, 2000). Thus, RACK1 appears to be associated with cellular proteins in a manner dependent or independent on the PKC activation.…”