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 ph osphodiesterase 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 -[3- (cyclopentoxy)-4-methoxyphenyl]-2-pyrrolidone} inhibited both PKA-phos phorylated HSPDE4D3 and the Ser-54 --> Asp mutant, with an IC50 value that was similar to 8-fold lower than that seen for the non-PKA-phosph orylated enzyme. Lower IC,, values for inhibition by rolipram were see n for a wide range of non-activated residue 54 mutants, except for tho se which had side-chains able to serve as hydrogen-bond donors, namely the Ser-54-->Thr, Ser-54-->Tyr and Ser-53-->Cys mutants. The G1u-53 - ->Ala mutant exhibited an activity comparable with that of the PKA pho sphorylated native enzyme and the Ser-54 --> Asp mutant but, in contra st to the native enzyme, was insensitive to activation by PKA, despite being more rapidly phosphorylated by this protein kinase. The activat ed Glu-53 -->Ala mutant exhibited a sensitivity to inhibition by rolip ram which was unchanged from that of the native enzyme. The double mut ant, Arg-51 --> Ala/Arg-52 --> Ala, showed no change in either enzyme activity or rolipram inhibition from the native enzyme and was incapab le of providing a substrate for PKA phosphorylation at Ser-54. No diff erence in inhibition by dipyridamole was seen for the native enzyme an d 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 t o involve disruption of an ion-pair interaction involving the negative ly charged Glu-53. The increase in susceptibility to inhibition by rol ipram upon PKA-mediated phosphorylation is suggested to involve the di sruption of a hydrogen-bond involving the side-chain hydroxy group of Ser-54.