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 (K(m) - 0.9 μM, and V(max) = 350 nmol/min/mg) and phosphoseryl LC₂₀ (K(m) = 2.6 μM, V(max) = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC₂₀ was inhibited by phosphoseryl LC₂₀ with an apparent K(i) of 2 μM. Compared to the reactions with phosphotyrosyl LC₂₀ as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher K(m) and lower V(max) values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher K(m) (23 μM), but a much higher V(max) (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.