The activity of calmodulin is altered by phosphorylation: modulation of calmodulin function by the site of phosphate incorporation

Abstract

Calmodulin transduces Ca2+ signals by binding to and activating essential regulatory enzymes. The large number of intracellular targets for calmodulin raises the possibility that mechanisms in addition to Ca2+ may modulate calmodulin activity. Phosphocalmodulin is found in cells and tissues, and calmodulin phosphorylation is enhanced by several mitogens. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreased its ability to activate Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). The major effect was a 2.5-fold increase in the concentration at which half-maximal velocity (K0.5) was attained, with no apparent alteration in the Vmax, or the K0.5 for Ca2+. In contrast, calmodulin phosphorylated on tyrosine residues by the insulin receptor kinase produced an increase in the Vmax, with no alteration in the affinity for CaM-kinase II or the K0.5 for Ca2+. Direct determination by surface plasmon resonance of the dissociation constants with a synthetic peptide corresponding to the calmodulin-binding domain of CaM-kinase II revealed that phosphorylation on serine/threonine residues of calmodulin significantly decreased its affinity for the peptide, while tyrosine phosphorylation had no effect on binding. In contrast to CaM-kinase II, neither serine/threonine nor tyrosine phosphorylation of calmodulin altered its ability to activate calcineurin. These data indicate that phosphorylation of calmodulin differentially modifies its interaction with individual target enzymes. Moreover, the amino acid residues phosphorylated provide an additional level of control. These results demonstrate that phosphorylation is an in vitro regulatory mechanism in the targeting of calmodulin responses and, coupled with the stoichiometric phosphorylation of calmodulin in rat hepatocytes, suggest that it may be relevant in intact cells.