CONSTRUCTION OF AN EPITOPE-TAGGED CALMODULIN USEFUL FOR THE ANALYSIS OF CALMODULIN-BINDING PROTEINS - ADDITION OF A HEMAGGLUTININ EPITOPE DOES NOT AFFECT CALMODULIN-DEPENDENT ACTIVATION OF SMOOTH-MUSCLE MYOSINLIGHT-CHAIN KINASE

An epitope-tagged calmodulin (CaM), capable of interacting with CaM-bi nding proteins in cellular extracts, would be a valuable tool for iden tifying proteins in signal transduction pathways involving calcium. A bacterial overexpression vector for epitope-tagged CaM has been constr ucted by inserting the coding sequence for a nine amino acid portion o f the influenza virus hemagglutinin (HA) protein into the initiation s ite of an overexpression vector for chicken CaM, The HA-CaM fusion pro duced in bacteria was compared to native CaM for its ability to activa te smooth muscle myosin light chain kinase (MLCK), one of the best und erstood CaM-dependent enzymes. MLCK activity was tested in both a puri fied system and a CaM-depleted ''native actomyosin'' preparation maint aining many of the regulatory properties of the intact smooth muscle. HA-CaM behaves identically to unmodified CaM in both systems, indicati ng that the RA epitope does not adversely affect CaM function. The rec ombinant HA-CaM was used to sensitively detect CaM interactions with s mooth muscle proteins in a modified gel overlay assay, using a monoclo nal antibody against the HA epitope as the secondary reagent. Enzymati cally active complexes of HA-CaM and MLCK could be immunoprecipitated from actomyosin preparations using the same monoclonal antibody and pr otein G-Sepharose beads. (C) 1997 Academic Press.