Abstract
Phosphodiesterase-5 (PDE5) is phosphorylated at a single serine residue by cyclic nucleotide-dependent protein kinases. To test for a direct effect of phosphorylation on the PDE5 catalytic site, independent of cGMP binding to the allosteric sites of the enzyme, binding of the catalytic site-specific substrate analog [3H]tadalafil to PDE5 was measured. Phosphorylation increased [3H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [3H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [3H]tadalafil exchange-dissociation rate from PDE5 more than 6-fold. Finally, phosphorylation increased affinity for hydrolysis of a catalytic site-specific cGMP analog, 2′-O-anthraniloyl-cGMP, by ∼3-fold. The combined results showed that phosphorylation activates PDE5 catalytic site independently of cGMP binding to the allosteric sites. The results suggested that phosphorylation acts in concert with allosteric cGMP binding to stimulate the PDE5 catalytic site, which should promote negative feedback regulation of the cGMP pathway in intact cells. By increasing the affinity of the catalytic site, phosphorylation should also consequently increase the potency and duration of PDE5 inhibitor action.
Footnotes
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This work was supported by National Institutes of Health Research Grants DK-40029 and DK-58277, National Institutes of Health Training Grant 5T32 HL-07751, and American Heart Association Postdoctoral Fellowship 032525B.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.133405.
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ABBREVIATIONS: PDE, phosphodiesterase; PKG, cGMP-dependent protein kinase; TLC, thin-layer chromatography; PAGE, polyacrylamide gel electrophoresis; PKA, cAMP-dependent protein kinase; KP, potassium phosphate; Ant, 2′-O-anthraniloyl.
- Received November 5, 2007.
- Accepted January 15, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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