Abstract
Myosin light chain kinase is a Ca2+/calmodulin-dependent protein kinase which exhibits a very high degree of protein substrate specificity. The regulatory light chain of myosin is the only known physiological substrate of the enzyme. Based upon epitope mapping of monoclonal antibodies which inhibit kinase activity competitively with respect to the light chain substrate, residues 235-319 of the rabbit skeletal muscle kinase have been proposed to contain a light chain-binding site (Herring, B. P., Stull, J. T., and Gallagher, P. J. (1990) J. Biol. Chem. 265, 1724-1730). With the expression of a truncated kinase, we have further localized this putative binding site to residues 235-294. Mutation of acidic residues at positions 269 and 270 of the kinase resulted in a 10-fold increase in the Km value for the myosin light chain, with no significant change in the Vmax value. In contrast, altering a cluster of acidic amino acids at positions 261-263 had little effect on the Km value for the myosin light chain. These results suggest that residues 269 and 270 may be involved in protein-substrate binding. Interestingly, these residues, located amino-terminal of the homologous catalytic core (positions 302-539), are in a region which is highly conserved among myosin light chain kinases, but not other protein kinases. It is probable that the homologous catalytic core contains structural elements required for phosphotransferase activity. The catalytic domain of myosin light chain kinase would therefore include these conserved elements together with additional specific substrate-binding residues.
Highlights
Acidic Residues Comprise Part of the Myosin Light Chain-binding Site on Skeletal Muscle Myosin Light Chain Kinase*
Altering a cluster of acidic amino acids at positions 261-263 had little effect on the K, value for the myosin light chain. These results suggest that residues 269 and 270 may be involved in protein-substrate binding. These residues, located amino-terminal of the homologous catalytic core, are in a region which is highly conserved among myosin light chain kinases, but not other protein kinases
Each of the inhibitory monoclonal antibodies reacted with this truncated kinase (Table I). This result further restricts the epitopes for these antibodies to residues 235-293 and indicates that the proposed myosin light chain-binding site is located amino-terminal of the catalytic core of the kinase
Summary
Protein Purification and Kinuse Assays-Rabbit skeletal muscle myosin light chain kinase was purified ai described previously (Nunnally et al, 1985) with the modifications of Herring et al (1990). COS cell lysates were prepared as described previously (Herring et al, 1990): Myosin light chain kinase activity, present in the COS cell lysates, was measured as “P incorporation into the rabbit skeletal muscle regulatory light chain. Lysates were diluted lOO-500-fold into the assay mixture Under these conditions, the activity of all the expressed myosin light chain kinaseswas completely Ca’+/calmodulin-dependent; in addition, lysates prepared from nontransfected. Mutagenesis-Oligonucleotide-directed mutagenesis was carried out as described by Zoller and Smith (1984)
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