Kennard A Grimes

Publications (5)20.54 Total impact

• Article: Phosphorylation increases affinity of the phosphodiesterase-5 catalytic site for tadalafil.

  Emmanuel P Bessay, Mitsi A Blount, Roya Zoraghi, Alfreda Beasley, Kennard A Grimes, Sharron H Francis, Jackie D Corbin
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  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 [(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.
  Journal of Pharmacology and Experimental Therapeutics 05/2008; 325(1):62-8. · 3.83 Impact Factor
• Article: Phosphorylation of phosphodiesterase-5 is promoted by a conformational change induced by sildenafil, vardenafil, or tadalafil.

  Emmanuel P Bessay, Roya Zoraghi, Mitsi A Blount, Kennard A Grimes, Alfreda Beasley, Sharron H Francis, Jackie D Corbin
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  ABSTRACT: Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, vardenafil, or tadalafil) or phosphorylation by cyclic nucleotide-dependent protein kinase causes an apparent conformational change in PDE5, as indicated by a shift in migration on non-denaturing PAGE gels and an altered pattern of tryptic digestion. Combination of cGMP and a PDE5 inhibitor or phosphorylation does not cause a further gel shift or change in tryptic digest. Phosphorylation of PDE5 is stimulated by inhibitors, and combination of cGMP and inhibitor does not cause further phosphorylation. Dephosphorylation of PDE5 by either purified phosphoprotein phosphatase-1 or -2A catalytic subunit or by a crude phosphatase mixture is not affected by cGMP or inhibitors, suggesting that phosphorylation itself maintains conformational exposure of the phosphorylation site. The combined results imply that cGMP binding to the catalytic site initiates negative feedback control of many cellular cGMP signaling pathways by directly stimulating phosphorylation and activation of PDE5; by exploiting this molecular mechanism, PDE5 inhibitors stimulate their own potencies.
  Frontiers in Bioscience 02/2007; 12:1899-910. · 3.52 Impact Factor
• Source

  Available from: Mitsi A Blount

  Article: Allosteric sites of phosphodiesterase-5 sequester cyclic GMP.

  Jun Kotera, Sharron H Francis, Kennard A Grimes, Alfreda Rouse, Mitsi A Blount, Jackie D Corbin
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  ABSTRACT: Phosphodiesterase-5 (PDE5) and cGMP-dependent protein kinase (PKG) play key roles in cGMP signaling. PDE5 has a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that binds cGMP at allosteric sites. We recently demonstrated that in corpus cavernosum, PDE5 concentration exceeds basal cGMP by ~5-fold making it possible that its allosteric sites could bind a significant fraction of the total cellular cGMP. It is hypothesized that the allosteric sites regulate cGMP signaling by sequestering cGMP. At 60 nM cGMP in vitro, which approaches a stimulated concentration of cGMP in rabbit corpus cavernosum, isolated R domain inhibits both cGMP hydrolysis by C domain and activation of PKG (IC50 values of 388 and 100 nM, respectively). Prior phosphorylation of R domain by cyclic nucleotide-dependent protein kinases, which increases its cGMP-binding affinity, also increases its potency for inhibiting both cGMP hydrolysis by C domain and cGMP activation of PKG (IC50 values of 58 and 38 nM, respectively). In rabbit corpus cavernosum, PDE5 concentration (94 nM) exceeds these values. These findings support our hypothesis that physiological concentrations of R domain regulate cGMP signaling by sequestering this nucleotide and that phosphorylation of R domain modulates this effect. This could provide for negative feedback control of cGMP-signaling.
  Frontiers in Bioscience 02/2004; 9:378-86. · 3.52 Impact Factor
• Source

  Available from: nih.gov

  Article: cGMP-dependent protein kinase protects cGMP from hydrolysis by phosphodiesterase-5.

  Jun Kotera, Kennard A Grimes, Jackie D Corbin, Sharron H Francis
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  ABSTRACT: The physiological effects of cGMP are largely determined by the activities of intracellular receptors, including cGMP-dependent protein kinase (PKG) and cGMP-binding cyclic nucleotide phosphodiesterases (PDEs), and the distribution of cGMP among these receptors dictates activity of the signalling pathway. In the present study, the effects of PKG-Ialpha or PKG-Ibeta on the rate of cGMP hydrolysis by the isolated PDE5 catalytic domain were examined. PKG-Ialpha strongly inhibited cGMP hydrolysis with an IC(50) value of 217 nM, which is similar to the physiological concentration of PKG in pig coronary artery reported previously. By contrast, PKG-Ibeta, which has lower affinity for cGMP than does PKG-Ialpha, inhibited cGMP hydrolysis with an IC(50) of approx. 1 microM. Inhibition by PKG-Ialpha was more effective than that by PKG-Ibeta, consistent with their relative affinities for cGMP. Autophosphorylation of PKGs increased their cGMP-binding affinities and their inhibitory effects on PDE5 hydrolysis of cGMP. Autophosphorylation of PKG-Ibeta increased its inhibitory potency on PDE5 hydrolysis of cGMP by 10-fold compared with a 2-fold increase upon autophosphorylation of PKG-Ialpha. The results indicate that cGMP bound to allosteric cGMP-binding sites of PKG is protected from hydrolysis by PDE5 and that persistent protection of cGMP by either non-phosphorylated or autophosphorylated PKGs may be a positive-feedback control to sustain cGMP signalling.
  Biochemical Journal 07/2003; 372(Pt 2):419-26. · 4.90 Impact Factor
• Article: Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity.

  Sharron H Francis, Emmanuel P Bessay, Jun Kotera, Kennard A Grimes, Li Liu, W Joseph Thompson, Jackie D Corbin
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  ABSTRACT: Substrate binding to the phosphodiesterase-5 (PDE5) catalytic site increases cGMP binding to the regulatory domain (R domain). The latter promotes PDE5 phosphorylation by cyclic nucleotide-dependent protein kinases, which activates catalysis, enhances allosteric cGMP binding, and causes PDE5A1 to apparently elongate. A human PDE5A1 R domain fragment (Val(46)-Glu(539)) containing the phosphorylation site (Ser(102)) and allosteric cGMP-binding sites was studied. The rate, cGMP dependence, and stoichiometry of phosphorylation of the PDE5 R domain by the catalytic subunit of cAMP-dependent protein kinase are comparable with that of the holoenzyme. Migration in native polyacrylamide gels suggests that either cGMP binding or phosphorylation produces distinct conformers of the R domain. Phosphorylation of the R domain increases affinity for cGMP approximately 10-fold (K(D) values 97.8 +/- 17 and 10.0 +/- 0.5 nm for unphospho- and phospho-R domains, respectively). [(3)H]cGMP dissociates from the phospho-R domain with a single rate (t(12) = 339 +/- 30 min) compared with the biphasic pattern of the unphospho-R domain (t(12) = 39.0 +/- 4.8 and 265 +/- 28 min, for the fast and slow components, respectively). Thus, cGMP-directed regulation of PDE5 phosphorylation and the resulting increase in cGMP binding affinity occur largely within the R domain. Conformational change(s) elicited by phosphorylation of the R domain within the PDE5 holoenzyme may also cause or participate in stimulating catalysis.
  Journal of Biological Chemistry 01/2003; 277(49):47581-7. · 4.77 Impact Factor