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.
Mesh Terms:
3',5'-Cyclic-AMP Phosphodiesterases, Animals, Antigens, Viral, COS Cells, Cell Line, Transformed, Cyclic AMP-Dependent Protein Kinases, Enzyme Activation, Epitopes, Humans, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Phosphodiesterase Inhibitors, Phosphorylation, Protein Conformation, Pyrrolidinones, Recombinant Proteins, Rolipram, Serine, Vesicular stomatitis Indiana virus
3',5'-Cyclic-AMP Phosphodiesterases, Animals, Antigens, Viral, COS Cells, Cell Line, Transformed, Cyclic AMP-Dependent Protein Kinases, Enzyme Activation, Epitopes, Humans, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Phosphodiesterase Inhibitors, Phosphorylation, Protein Conformation, Pyrrolidinones, Recombinant Proteins, Rolipram, Serine, Vesicular stomatitis Indiana virus
Biochem. J.
Date: Jul. 01, 1998
PubMed ID: 9639573
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