ACTIVATION OF CALCINEURIN AND SMOOTH-MUSCLE MYOSIN LIGHT-CHAIN KINASEBY MET-TO-LEU MUTANTS OF CALMODULIN

The effects of replacement of each of the individual Met in calmodulin (CaM) with Leu on the activation of two CaM target enzymes [smooth mu scle myosin light chain kinase (smMLCK) and calcineurin (CN)] were inv estigated. The K-D and P-max (percentage maximal activation) values fo r activation of both enzymes by M76L-CaM were indistinguishable from w ild-type (wt)-CaM, which is consistent with the location of Met-76 in the central linker that is not involved in target protein interaction. The other eight Met in CaM are exposed in the hydrophobic surfaces th at are involved in target-enzymes binding, and in general equivalent e ffects are observed for substitutions of Leu for Met residues in homol ogous positions in the two CaM domains. However, the importance of the interaction of specific Met residues with the target enzyme depends o n the particular enzyme. Leu substitution at Met-36 or Met-109 reduced the affinity of MLCK for the mutant and the maximal activation of CN. MLCK had a higher K-D for M51L-CaM whereas M124L-CaM activated the ki nase to only 68% of maximal activity induced by wt-CaM; these mutants were indistinguishable from wt-CaM in activation of CN, M71L- and M144 L-CaMs behaved like wt-CaM in activation of MLCK, but activated the ph osphatase to only about 80% of maximal activity induced by wt-CAM. M72 L-CaM exhibited an increased affinity for MLCK compared to wt-CaM and slightly decreased maximal activation, whereas M145L-CaM exhibited max imal activation significantly greater than that due to wt-CaM; these m utants behaved like wt-CaM with respect to CN activation. Finally, a m utant CaM in which all four C-terminal Met were replaced by Leu (M-4-C T-L-4-CaM) had similar affinities for MLCK and CN as wt-CaM but maxima l activation of these enzymes by this mutant was only 60-70 % of that achieved with wt-CaM. These results imply that, in addition to removin g the autoinhibitory domain from the active site of the target enzyme, CaM must induce a conformational change in the active site itself.