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 myosin light chain kinase.
An epitope-tagged calmodulin (CaM), capable of interacting with CaM-binding proteins in cellular extracts, would be a valuable tool for identifying proteins in signal transduction pathways involving calcium. A bacterial overexpression vector for epitope-tagged CaM has been constructed by inserting the coding sequence for a nine amino acid portion of the influenza virus hemagglutinin (HA) protein into the initiation site of an overexpression vector for chicken CaM. The HA-CaM fusion produced in bacteria was compared to native CaM for its ability to activate smooth muscle myosin light chain kinase (MLCK), one of the best understood CaM-dependent enzymes. MLCK activity was tested in both a purified system and a CaM-depleted "native actomyosin" preparation maintaining many of the regulatory properties of the intact smooth muscle. HA-CaM behaves identically to unmodified CaM in both systems, indicating that the HA epitope does not adversely affect CaM function. The recombinant HA-CaM was used to sensitively detect CaM interactions with smooth muscle proteins in a modified gel overlay assay, using a monoclonal antibody against the HA epitope as the secondary reagent. Enzymatically active complexes of HA-CaM and MLCK could be immunoprecipitated from actomyosin preparations using the same monoclonal antibody and protein G-Sepharose beads.
Szymanska
G
G
Department of Biology, Boston College, Chestnut Hill, Massachusetts 02167-3811, USA.
O'Connor
M B
MB
O'Connor
C M
CM
D000205:Actomyosin / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000235:genetics; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002645:Chickens; D004586:Electrophoresis / Q000379:methods; D004789:Enzyme Activation; D000939:Epitopes; D005822:Genetic Vectors / Q000235:genetics; D006388:Hemagglutinins / Q000276:immunology*; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D000939:Epitopes; D006388:Hemagglutinins; D018994:Myosin Light Chains; D011993:Recombinant Fusion Proteins; D000205:Actomyosin; D009219:Myosin-Light-Chain Kinase; D002118:Calcium