Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding do mains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE, plasma membrane (PM) Ca2+-A TPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal ni tric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kin ase (CaM kinase II). In general, tyrosine phosphorylation led to an increas e in the activatory properties of calmodulin (CaM). For plasma membrane (PM ) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (K-act). In c ontrast, for calcineurin and NOS phosphorylation of CaM significantly incre ased the V-max. For MLCK, however, neither V-max nor K-act were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylatio n of Tyr99 on CaM generally increased its affinity for the peptides.