Phosphorylation of phosphodiesterase‐5 by cyclic nucleotide‐dependent protein kinase alters its catalytic and allosteric cGMP‐binding activities

Abstract

In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem. 265, 14971±14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50±70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2±0.5 mm PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the K m value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 mm. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed.

Mechanisms of action of PDE5 inhibition in erectile dysfunction

“…Phosphorylation of PDE5 by protein kinase G (PKG) augments the enzymatic activity as well as the affinity of PDE5 allosteric sites for cGMP. 14,15 The level of enzymatic activity has been shown to increase in parallel with phosphorylation, and the increase in activity is typically about 1.6-fold.…”

Characteristics of Photoreceptor PDE (PDE6): similarities and differences to PDE5

“…3 For PDE5, enzyme activation most likely proceeds via phosphorylation of the catalytic subunits and allosteric changes in cGMP binding to the GAF domains. 20,21 (While PDE5 has been reported to copurify with the PDE6 g subunit, 22 the physiological significance is uncertain because the g subunit lacks binding or inhibitory activity toward PDE5 in vitro. 23 )…”

“…20,35,36 The g subunit of PDE6 also acts as a substrate for phosphorylation at several distinct sites within the central region of this 10 kDa protein. Phosphorylation of g at Thr (22) or Thr (35) has little effect on the PDE6 holoenzyme itself, but greatly diminishes the ability of activated transducin to bind the g subunit and relieve inhibition of catalysis.…”

Single step isolation of sildenafil from commercially available Viagra™ tablets

“…[7][8][9] Physical properties of this preparation were similar to those of a purified sample from Pfizer. Molecular weight of sildenafil (474 g/mol) was confirmed by positive ion nanospray and MALDI mass spectrometry.…”

“…To date, most research has focused on the effect of sildenafil on vascular events in penile corpora cavernosa, but there is increased interest in potential efficacy of sildenafil in modulating cyclic nucleotide signaling in other tissues. [4][5][6][7][8][9][10][11] In addition, biochemical characteristics of the interaction of sildenafil with PDE5 and perhaps other proteins are still poorly understood. Investigation of these basic processes is compromised by the fact that purified sildenafil is not commercially available.…”