Phosphorylation of the minor sites of CaD22 reduced the inhibition of actomyosin ATPase activity. Actin-activated S1-ATPase as a function of phosphorylated and unphosphorylated CaD22WT (circles), CaD22DD (triangles up), and CaD22AA (triangles down) is shown. Open symbols show the effect of unphosphorylated CaD22, whereas solid symbols show the corresponding species after 20 h treatment with PAK. The inset shows actin-activated S1-ATPase as a function of the concentrations of CaD35WT and CaD35DD. Actin, smooth muscle tropomyosin, and myosin S1 were 10 μM, 2.2 μM, and 0.1 μM, respectively. The final solution composition was 1 mM ATP, 3 mM MgCl2, 31 mM NaCl, 10 mM imidazole pH 7.0, at 25°C.