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 ∼ 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.

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Author notes

1

Present address: BioFrontera Pharmaceuticals GmbH, GIZ Hemmelratherweg 201 51377, Leverkusen, Germany.

2

Present address: Department of Biochemistry, University of Sheffield, Sheffield, U.K.

3

These two authors contributed equally to this work.