Attenuation of the activity of the cAMP-specific phosphodiesterase PDE4A5 by interaction with the immunophilin XAP2.
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 approximately 60%, with an IC50 of 120 nm, and reduced the IC50 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 IC50 for rolipram. Native XAP2 and PDE4A5 could be co-immunoprecipitated from the brain. The isolated COOH-terminal half of XAP2 (amino acids 170-330), containing its tetratricopeptide repeat domain, but not the isolated NH2-terminal half (amino acids 1-169), containing the immunophilin homology region, similarly reduced PDE4A5 activity and its IC50 for rolipram. Mutation of Arg271 to alanine, in the XAP2 tetratricopeptide repeat region, attenuated its ability to both interact with PDE4A5 in two-hybrid assays and to inhibit PDE4A5 activity. Either the deletion of a specific portion of the unique amino-terminal 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.
Bolger
Graeme B
GB
Veterans Affairs Medical Center, Huntsman Cancer Institute, Department of Medicine, Division of Oncology, University of Utah Health Sciences Center, Salt Lake City, Utah 84148, USA. Graeme.Bolger@ccc.uab.edu
Peden
Alexander H
AH
Steele
Michael R
MR
MacKenzie
Carolynn
C
McEwan
David G
DG
Wallace
Derek A
DA
Huston
Elaine
E
Baillie
George S
GS
Houslay
Miles D
MD
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020889:Rolipram / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D020033:Protein Isoforms; D011506:Proteins; D011993:Recombinant Fusion Proteins; C103265:aryl hydrocarbon receptor-interacting protein; D005576:Colforsin; D000242:Cyclic AMP; D005982:Glutathione Transferase; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517355:PDE4A protein, human; D020889:Rolipram; D000409:Alanine; D015056:1-Methyl-3-isobutylxanthine