PHOSPHORYLATION OF CALMODULIN ALTERS ITS POTENCY AS AN ACTIVATOR OF TARGET ENZYMES

Previous work has shown that calmodulin (CaM) is constitutively phosph orylated in rat liver, probably by casein kinase II [Quadroni, M., Jam es, P., and Carafoli, E. (1994) J. Biol. Chem. 269, 16116-16122]. A pr ocedure is now described for the isolation of the phosphorylated forms of calmodulin (PCaM) free from CaM, since in vitro phosphorylation ex periments yield a 50:50 mixture of 3-4 times phosphorylated CaM and na tive CaM. The activation of six target enzymes by PCaM was tested: myo sin light chain kinase, 3',5'-cyclic nucleotide phosphodiesterase, pla sma membrane Ca2+-ATPase, Ca2+-CaM-dependent protein phosphatase 2B (c alcineurin), neuronal nitric oxide synthase, and CaM-kinase II. In gen eral, the phosphorylation of CaM caused a decrease in enzyme binding a ffinity, increasing the K-act by 2-4-fold for MLCK, PDE, PM Ca2+-ATPas e, and calcineurin. The V-max at saturating concentrations of PCaM was less affected, with the exception of CaM-kinase II, which was only mi nimally activated by PCaM and NOS whose V-max was increased 2.6 times by PCaM with respect to CaM. Phosphorylation of calmodulin had very li ttle effect on the binding of calcium to the enzyme despite the fact t hat Ser 101 which is phosphorylated is located in the third calcium bi nding loop. CD measurements performed on CaM and PCaM indicated that p hosphorylation causes a marked decrease in the a-helical content of th e protein. Phosphorylated CaM is very prone to dephosphorylation and w as thus tested as a substrate for several phosphatases. It was unaffec ted by calcineurin (PP2B), but was a reasonable substrate for the plei otropic phosphatases PP1 gamma and PP2A.