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

Eur J Biochem. 2000 May;267(9):2760-7. doi: 10.1046/j.1432-1327.2000.01297.x.

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 microM 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 Km 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 microM. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3',5'-Cyclic-GMP Phosphodiesterases
  • Allosteric Regulation
  • Animals
  • Catalysis
  • Cattle
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Cyclic GMP / metabolism*
  • Cyclic GMP-Dependent Protein Kinases
  • Cyclic Nucleotide Phosphodiesterases, Type 5
  • Phosphoric Diester Hydrolases / metabolism*
  • Phosphorylation
  • Protein Binding
  • Protein Kinases / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Recombinant Proteins / metabolism

Substances

  • Recombinant Proteins
  • Protein Kinases
  • Cyclic AMP-Dependent Protein Kinases
  • Cyclic GMP-Dependent Protein Kinases
  • Phosphoric Diester Hydrolases
  • 3',5'-Cyclic-GMP Phosphodiesterases
  • Cyclic Nucleotide Phosphodiesterases, Type 5
  • Pde5a protein, rat
  • Cyclic GMP