GAIN OF FUNCTION MUTATIONS FOR YEAST CALMODULIN AND CALCIUM-DEPENDENTREGULATION OF PROTEIN-KINASE ACTIVITY

Yeast calmodulin binds only three calcium ions in the presence of mill imolar concentrations of magnesium due to a defective calcium-binding sequence in its carboxyl terminal domain. Yeast calmodulin's diminishe d calcium-binding activity can be restored to that of other calmodulin s by the use of site-directed mutagenesis to substitute its fourth cal cium-binding domain with that of a vertebrate calmodulin sequence. How ever, the repair of yeast calmodulin's calcium-binding activity is not sufficient to repair quantitatively yeast calmodulin's defective prot ein kinase activator activity. Yeast calmodulin's activator activity w ith smooth muscle and skeletal muscle myosin light chain kinases and b rain calmodulin-dependent protein kinase II can be progressively repai red by additional substitutions of vertebrate calmodulin sequences, pr ovided that the four calcium-binding sites remain intact. An unexpecte d result obtained during the course of these studies was the observati on that myosin light chain kinases from smooth and skeletal muscle tis sues can respond differently to mutations in calmodulin. These and pre vious results indicate that the binding of four calcium ions by calmod ulin is necessary but not sufficient to bring about quantitative activ ation of protein kinases, and are consistent with the conformational s election/restriction model of the dynamic equilibrium among calcium, c almodulin and each calmodulin regulated enzyme.