Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase.
Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D005459:Fluorides / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology; D009218:Myosins / Q000378:metabolism; D009532:Nickel / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010768:Phosphoserine / Q000378:metabolism; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases; D012694:Serine / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014638:Vanadates; D014639:Vanadium / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D002147:Calmodulin; D014639:Vanadium; D010768:Phosphoserine; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D008345:Manganese; D012694:Serine; D009532:Nickel; D010749:Phosphoprotein Phosphatases; D010744:Phosphoric Monoester Hydrolases; C022394:phosphoserine phosphatase; D017027:Protein Tyrosine Phosphatases; D009218:Myosins; D015032:Zinc; D005459:Fluorides