title: Substrate- and kinase-directed regulation of phosphorylation of a cGMP-binding phosphodiesterase by cGMP.
abstract: A purified bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) was rapidly phosphorylated by purified bovine lung cGMP-dependent protein kinase (cGK). Within a physiological concentration range, cGK catalyzed phosphorylation of cG-BPDE at a rate approximately 10 times greater than did equimolar concentrations of purified catalytic subunit of cAMP-dependent protein kinase (cAK). cG-BPDE was a poor substrate for either purified protein kinase C or Ca2+/calmodulin-dependent protein kinase II. Binding of cGMP to the cG-BPDE binding site was required for phosphorylation since (a) phosphorylation of cG-BPDE by the catalytic subunit of cAK was cGMP-dependent, (b) phosphorylation of cG-BPDE in the presence of a cGMP analog specific for activation of cGK was cGMP-dependent, and (c) occupation of the cG-BPDE hydrolytic site with competitive inhibitors did not produce the cGMP-dependent effect. cGMP-dependent phosphorylation of cG-BPDE by both cGK and cAK occurred at serine. Proteolytic digestion of cG-BPDE phosphorylated by either cGK or cAK revealed the same phosphopeptide pattern, suggesting that phosphorylation by the two kinases occurred at the same or adjacent site(s). Tryptic digestion of cG-BPDE phosphorylated by cGK and [gamma-32P]ATP produced a single major phosphopeptide of approximately 2 kDa with the following amino-terminal sequence: Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg- Radioactivity was released during the third cycle of Edman degradation. cG-BPDE is one of few specific in vitro cGK substrates of known function to be identified. Elevation of intracellular cGMP may cause phosphorylation of cG-BPDE by modulating the substrate site availability as well as by activating cGK. Such regulation would greatly increase the selectivity of the phosphorylation of cG-BPDE and would represent a unique mechanism of action of a cyclic nucleotide or other second messenger.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615.
authors.forename: M K
authors.initials: MK
authors.lastname: Thomas
authors.forename: S H
authors.initials: SH
authors.lastname: Francis
authors.forename: J D
authors.initials: JD
authors.lastname: Corbin
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
chemical_list: D046911:Macromolecular Substances; D010748:Phosphopeptides; D011494:Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP