Attenuation of the Activity of the cAMP-specific Phosphodiesterase PDE4A5 by Interaction with the Immunophilin XAP2

Abstract

The cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4A5 interacted with the immunophilin XAP2 in a yeast two-hybrid assay. The interaction was confirmed in biochemical pull-down analyses. The interaction was specific, in that PDE4A5 did not interact with the closely related immunophilins AIPL1, FKBP51, or FKBP52. XAP2 also did not interact with other PDE4A isoforms or typical isoforms from the three other PDE4 subfamilies. Functionally, XAP2 reversibly inhibited the enzymatic activity of PDE4A5, increased the sensitivity of PDE4A5 to inhibition by the prototypical PDE4 inhibitor 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and attenuated the ability of cAMP-dependent protein kinase to phosphorylate PDE4A5 in intact cells. XAP2 maximally inhibited PDE4A5 by ϳ60%, with an IC 50 of 120 nM, and reduced the IC 50 for rolipram from 390 nM to 70 -90 nM. Co-expression of XAP2 and PDE4A5 in COS7 cells showed that they could be co-immunoprecipitated and also reduced both the enzymatic activity of PDE4A5 and its IC 50 for rolipram. Native XAP2 and PDE4A5 could be coimmunoprecipitated from the brain. The isolated COOHterminal half of XAP2 (amino acids 170 -330), containing its tetratricopeptide repeat domain, but not the isolated NH 2 -terminal half (amino acids 1-169), containing the immunophilin homology region, similarly reduced PDE4A5 activity and its IC 50 for rolipram. Mutation of Arg 271 to alanine, in the XAP2 tetratricopeptide repeat region, attenuated its ability to both interact with PDE4A5 in twohybrid assays and to inhibit PDE4A5 activity. Either the deletion of a specific portion of the unique aminoterminal region or specific mutations in the regulatory UCR2 domain of PDE4A5 attenuated its ability be inhibited by XAP2. We suggest that XAP2 functionally interacts with PDE4A5 in cells.Signal transduction mediated by the second messenger cAMP is pivotal in a multitude of cellular processes, including the action of numerous hormones, neurotransmitters, and growth factors (1). The PDE4 1 cAMP-specific phosphodiesterases modulate cAMP signaling by their ability to hydrolyze cAMP and thereby contribute to the regulation of its levels in cells (2, 3). PDE4 enzymes can be differentiated from other cyclic nucleotide phosphodiesterases by sequence homology in the catalytic region of the proteins and by the presence of unique regions of amino acid sequence in the amino-terminal half of the proteins, outside the catalytic region, which are called upstream conserved regions 1 and 2 (UCR1 and UCR2) (2, 4). In addition, PDE4s are also characterized by their ability to be inhibited by a specific class of drugs, such as rolipram, which have anti-depressant, anti-inflammatory, and smooth muscle relaxant activity in humans (2, 3). At least 15 different PDE4 isoforms have been described in mammals, which are encoded by four different genes (PDE4A, PDE4B, PDE4C, and PDE4D), with additional diversity being generated by the generation of alternatively spliced mRNAs from each gene (2, 3).PDE4A5 is a cAMP-s...

Phosphodiesterases link the aryl hydrocarbon receptor complex to cyclic nucleotide signaling

“…Recently, type 2A and type 4A5 phosphodiesterases (PDE) were identified as new binding partners of XAP2 providing an unexpected molecular link between the AHR and the cyclic nucleotide signaling system [57,58]. From this point we shall describe some important new aspects of cyclic nucleotide signaling including main cellular functions and crosstalk with the AHR pathway.…”

“…Bolger et al [57] observed for the first time the interaction of a type 4 PDE with XAP2. In their work the authors showed that specifically the PDE4A5 isoform could bind XAP2.…”

“…The PDE4A gene is expressed in a wide variety of tissues including the lung, various regions of the brain and in leukocytes [57,84,85]. Membrane as well as cytosolic distribution of PDE4A proteins has been observed [82].…”

“…Bolger et al [57] identified XAP2 as a specific binding partner for the cAMP-specific PDE4A5. This interaction was also mediated by the TPR domain containing C-terminus region of XAP2 and it seems to be specifically restricted to the A5 isoform of PDE4, since PDE4 isoforms A1, A8, B2, B3, D3, D4, and D5 did not bind XAP2.…”

“…This interaction was also mediated by the TPR domain containing C-terminus region of XAP2 and it seems to be specifically restricted to the A5 isoform of PDE4, since PDE4 isoforms A1, A8, B2, B3, D3, D4, and D5 did not bind XAP2. A careful mapping study revealed that the amino-terminal region of PDE4A5 holding an Glu-Glu-Leu-Asp (EELD) amino acid sequence motif in the upstream conserved region-2 exposes the major binding site of XAP2 [57]. This segment has no apparent similarity to GAF-B of PDE2A, suggesting different modes of XAP2 binding among PDE isotypes.…”