Functional role of the C-terminal domain of smooth muscle myosin light chain kinase on the phosphorylation of smooth muscle myosin.
Smooth muscle myosin light chain kinase (MLCK) is known to bind to thin filaments and myosin filaments. Telokin, an independently expressed protein with an identical amino acid sequence to that of the C-terminal domain of MLCK, has been shown to bind to unphosphorylated smooth muscle myosin. Thus, the functional significance of the C-terminal domain and the molecular morphology of MLCK were examined in detail. The C-terminal domain was removed from MLCK by alpha-chymotryptic digestion, and the activity of the digested MLCK was measured using myosin or the isolated 20-kDa light chain (LC20) as a substrate. The results showed that the digestion increased K(m) for myosin 3-fold whereas it did not change the value for LC20. In addition, telokin inhibited the phosphorylation of myosin by MLCK by increasing K(m) but only slightly increased K(m) for LC20. Electron microscopy indicated that MLCK was an elongated molecule but was flexible so as to form folded conformations. MLCK was crosslinked to unphosphorylated heavy meromyosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the absence of Ca(2+)/calmodulin (CaM), and electron microscopic observation of the products revealed that the MLCK molecule bound to the head-tail junction of heavy meromyosin. These results suggest that MLCK binds to the head-tail junction of unphosphorylated myosin through its C-terminal domain, where LC20 can be promptly phosphorylated through its catalytic domain following the Ca(2+)/CaM-dependent activation.
Numata
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Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
Katoh
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Yazawa
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D000818:Animals; D015153:Blotting, Western; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D009124:Muscle Proteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000201:enzymology* / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D009218:Myosins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides; D010766:Phosphorylation; D018583:Pliability; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D009124:Muscle Proteins; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D010446:Peptide Fragments; D010455:Peptides; C060640:telokin; D009219:Myosin-Light-Chain Kinase; D002918:Chymotrypsin; D009218:Myosins