Differential regulation of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isoenzymes by cyclic AMP-dependent protein kinase and calmodulin-dependent phosphatase.
Purified bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) contains isozymes that are composed of two distinct subunits with molecular masses of 60,000 and 63,000 daltons. Analysis by NaDodSO4 gel electrophoresis and autoradiography of a phosphodiesterase sample phosphorylated in the presence of [32P]ATP and bovine heart cAMP-dependent protein kinase catalytic subunit revealed that only the 60-kDa subunit was phosphorylated. By using an isozyme preparation greatly enriched with the 60-kDa subunit, the following observations regarding the subunit phosphorylation were made. First, the phosphorylation resulted in the maximal incorporation of about 2 mol of phosphate per mol of subunit. Second, complete inhibition of 60-kDa subunit phosphorylation was approached at a saturating concentration of Ca2+ when a molar ratio of calmodulin to phosphodiesterase of 2:1 was used. No inhibition was observed in the presence of either Ca2+ or calmodulin alone. Third, the phosphorylation was accompanied by a decrease in the enzyme affinity for calmodulin; calmodulin concentrations required for 50% activation of nonphosphorylated and maximally phosphorylated phosphodiesterase isozyme samples were 0.51 and 9.3 nM, respectively. Fourth, the phosphodiesterase isozyme could be dephosphorylated by the calmodulin-dependent phosphatase (calcineurin) in the presence of Ni2+ or Mn2+, the dephosphorylation being associated with an increase in the enzyme affinity for calmodulin. Fifth, peak II rabbit liver phosphoprotein phosphatase catalytic unit did not catalyze the dephosphorylation of the phosphodiesterase isozyme.
Sharma
R K
RK
Wang
J H
JH
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology*; D002148:Calmodulin-Binding Proteins; D002417:Cattle; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007527:Isoenzymes; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D002118:Calcium