Bessay Emmanuel P, Blount Mitsi A, Zoraghi Roya, Beasley Alfreda, Grimes Kennard A, Francis Sharron H, Corbin Jackie D
Department of Molecular Physiology and Biophysics, 702 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232-0615, USA.
J Pharmacol Exp Ther. 2008 Apr;325(1):62-8. doi: 10.1124/jpet.107.133405. Epub 2008 Jan 16.
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 [(3)H]tadalafil to PDE5 was measured. Phosphorylation increased [(3)H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [(3)H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [(3)H]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 approximately 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.
磷酸二酯酶-5（PDE5）可被环核苷酸依赖性蛋白激酶在单个丝氨酸残基上磷酸化。为了测试磷酸化对PDE5催化位点的直接作用，在不依赖于cGMP与该酶变构位点结合的情况下，测定了催化位点特异性底物类似物[（3）H]他达拉非与PDE5的结合。磷酸化使[（3）H]他达拉非的结合增加了3倍，而cGMP则使其增加了1.6倍。两种处理方式的联合使[（3）H]他达拉非的结合增加了4倍以上，且仅在次最大刺激时效应呈相加性。与亲和力增加一致，磷酸化使[（3）H]他达拉非从PDE5上的交换解离速率减慢了6倍以上。最后，磷酸化使对催化位点特异性cGMP类似物2'-O-邻氨基苯甲酰基-cGMP水解的亲和力增加了约3倍。综合结果表明，磷酸化可独立于cGMP与变构位点的结合而激活PDE5催化位点。结果提示，磷酸化与变构性cGMP结合协同作用以刺激PDE5催化位点，这应能促进完整细胞中cGMP途径的负反馈调节。通过增加催化位点的亲和力，磷酸化还应相应增加PDE5抑制剂作用的效力和持续时间。
