Abstract
Catalytic cores of skeletal and smooth muscle myosin light chain kinases and Ca2+/calmodulin-dependent protein kinase II are regulated intrasterically by different regulatory segments containing autoinhibitory and calmodulin-binding sequences. The functional properties of these regulatory segments were examined in chimeric kinases containing either the catalytic core of skeletal muscle myosin light chain kinase or Ca2+/calmodulin-dependent protein kinase II with different regulatory segments. Recognition of protein substrates by the catalytic core of skeletal muscle myosin light chain kinase was altered with the regulatory segment of protein kinase II but not with smooth muscle myosin light chain kinase. Similarly, the catalytic properties of the protein kinase II were altered with regulatory segments from either myosin light chain kinase. All chimeric kinases were dependent on Ca2+/calmodulin for activity. The apparent Ca2+/calmodulin activation constant was similarly low with all chimeras containing the skeletal muscle catalytic core. The activation constant was greater with chimeric kinases containing the catalytic core of Ca2+/calmodulin-dependent protein kinase II with its endogenous or myosin light chain kinase regulatory segments. Thus, heterologous regulatory segments affect substrate recognition and kinase activity. Furthermore, the sensitivity to calmodulin activation is determined primarily by the respective catalytic cores, not the calmodulin-binding sequences.
Highlights
The crystal structures of Ca2ϩ/calmodulin-dependent protein kinase I [12] and twitchin kinase [13, 14] presented structural insights into the autoinhibitory mechanism
Constructs of Wild-type, Truncated, and Chimeric Kinases—All of the wild-type, truncated, and chimeric kinases were constructed as shown in Fig. 1. tSkMLCK was made as described previously [9] by removing the N-terminal 256 amino acid residues, which have no known functional role. tCaMKII was constructed by deleting the C-terminal association domain responsible for oligomerization of the holoenzyme [30, 31] The calmodulin-binding sequence and additional N-terminal residues of neuronal nitric oxide synthase were used to construct the chimera tSkMLCK[nNOS]
A NcoI restriction site was created between the catalytic core and the regulatory segment of the myosin light chain kinases, CaMKII, and the N termini of the calmodulin-binding sequence of nitric oxide synthase
Summary
Expression and Purification of Recombinant Myosin Light Chain and Calmodulin—Recombinant human smooth and rabbit skeletal muscle myosin light chains were expressed and purified as described previlino)propanesulfonic acid; NOS, nitric oxide synthase. Stability Studies on Chimeric Kinases—For limited proteolysis, COS cell lysates containing kinases expressed at approximately 5–10 ng/l were diluted 1:5 into the following 50-l reaction mixture: 6 mM MOPS, pH 7.5, 0.6 mM dithiothreitol, and 1 M calmodulin in the presence of 1 mM CaCl2 or 5 mM EGTA. An aliquot of each digest was diluted into SDS sample buffer, and the resultant proteolytic digestion pattern examined following SDS-polyacrylamide gel electrophoresis and immunoblotting [5] In another aliquot, myosin light chain kinase activity was measured at room temperature following 4 –12-fold dilution of the proteolyzed extracts into reaction mixtures containing either Ca2ϩ or EGTA as described above. Immunoblotting following SDS-polyacrylamide gel electrophoresis was performed as described above
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