cAMP-specific phosphodiesterase HSPDE4D3 mutants which mimic activation and changes in rolipram inhibition triggered by protein kinase A phosphorylation of Ser-54: generation of a molecular model.
Ser-13 and Ser-54 were shown to provide the sole sites for the protein kinase A (PKA)-mediated phosphorylation of the human cAMP-specific phosphodiesterase isoform HSPDE4D3. The ability of PKA to phosphorylate and activate HSPDE4D3 was mimicked by replacing Ser-54 with either of the negatively charged amino acids, aspartate or glutamate, within the consensus motif of RRES54. The PDE4 selective inhibitor rolipram ¿4-[3-(cyclopentoxy)-4-methoxyphenyl]-2-pyrrolidone¿ inhibited both PKA-phosphorylated HSPDE4D3 and the Ser-54-->Asp mutant, with an IC50 value that was approximately 8-fold lower than that seen for the non-PKA-phosphorylated enzyme. Lower IC50 values for inhibition by rolipram were seen for a wide range of non-activated residue 54 mutants, except for those which had side-chains able to serve as hydrogen-bond donors, namely the Ser-54-->Thr, Ser-54-->Tyr and Ser-54-->Cys mutants. The Glu-53-->Ala mutant exhibited an activity comparable with that of the PKA phosphorylated native enzyme and the Ser-54-->Asp mutant but, in contrast to the native enzyme, was insensitive to activation by PKA, despite being more rapidly phosphorylated by this protein kinase. The activated Glu-53-->Ala mutant exhibited a sensitivity to inhibition by rolipram which was unchanged from that of the native enzyme. The double mutant, Arg-51-->Ala/Arg-52-->Ala, showed no change in either enzyme activity or rolipram inhibition from the native enzyme and was incapable of providing a substrate for PKA phosphorylation at Ser-54. No difference in inhibition by dipyridamole was seen for the native enzyme and the Ser-54-->Asp and Ser-54-->Ala mutants. A model is proposed which envisages that phosphorylation by PKA triggers at least two distinct conformational changes in HSPDE4D3; one of these gives rise to enzyme activation and another enhances sensitivity to inhibition by rolipram. Activation of HSPDE4D3 by PKA-mediated phosphorylation is suggested to involve disruption of an ion-pair interaction involving the negatively charged Glu-53. The increase in susceptibility to inhibition by rolipram upon PKA-mediated phosphorylation is suggested to involve the disruption of a hydrogen-bond involving the side-chain hydroxy group of Ser-54.
Hoffmann
R
R
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Davidson and Wolfson Buildings, IBLS, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Wilkinson
I R
IR
McCallum
J F
JF
Engels
P
P
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology; D019556:COS Cells; D002461:Cell Line, Transformed; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010766:Phosphorylation; D011487:Protein Conformation; D011760:Pyrrolidinones / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020889:Rolipram; D012694:Serine / Q000378:metabolism*; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000276:immunology
D000956:Antigens, Viral; D000939:Epitopes; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D011994:Recombinant Proteins; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D020889:Rolipram