CHARACTERIZATION OF THE COOH TERMINUS OF NONMUSCLE CALDESMON MUTANTS LACKING MITOSIS-SPECIFIC PHOSPHORYLATION SITES

Phosphorylation of rat non muscle caldesmon by cdc2 kinase causes redu ction in most of caldesmon's properties, including caldesmon's binding to actin, myosin, and calmodulin, as well as its inhibition of actomy osin ATPase, We have generated and characterized the COOH terminus of caldesmon mutants lacking mitosis-specific phosphorylation sites, beca use the COOH-terminal half of caldesmon contains all ? putative Ser or Thr sites for cdc2 kinase. Codons for the 7 putative Ser or Thr resid ues have been mutated to Ala, and resultant mutants were bacterially e xpressed. Analyses of the phosphopeptide maps of these mutants have id entified 6 sites, including Ser-249, Ser-462, Thr-468, Ser-491, Ser-49 7, and Ser-527 as the mitosis-specific phosphorylation sites, whereas the phosphorylation of the remaining site, Thr-377, is not detected by this assay method. Actin binding experiments have suggested that 5 si tes including Ser-249, Ser-462, Thr-468, Ser-491, and Ser-497 are impo rtant for the phosphorylation-dependent reduction in actin binding. Ch aracterization of a mutant lacking all 7 Ser or Thr sites (7-fold muta nt) has revealed that 7-fold mutation eliminates all phosphorylation s ites by cdc2 kinase, While the in vitro properties of the 7-fold mutan t, including actin, myosin, and calmodulin binding and inhibition of a ctomyosin ATPase, are very similar to those of nonmutated protein, suc h properties are not affected by the treatment with cdc2 kinase in con trast to nonmutated protein. This mutant should thus be useful to expl ore the functions of the mitosis-specific phosphorylation of caldesmon .