Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis.
Site-directed mutagenesis of smooth muscle myosin light chain kinase was applied to define its autoinhibitory domain. Mutants were all initiated at Leu-447 but contained varying lengths of C-terminal sequence. Those containing the complete C-terminal sequence to Glu-972 possessed kinase activities that were calmodulin-dependent. Removal of the putative inhibitory domain by truncation to Thr-778 resulted in generation of a constitutively active (calmodulin-independent) species. Thus, the inhibitory domain lies to the C-terminal side of Thr-778. Truncation to Lys-793 and to Trp-800 also resulted in constitutively active mutants, although the specific activity of the latter was less than the other mutants. None of the truncated mutants bound calmodulin. For each mutant, the Km values with respect to ATP and to the 20,000-dalton light chain were similar to values obtained with the native enzyme. The presence of the inhibitory domain was detected by activation of kinase activity following limited proteolysis with trypsin. Using this procedure, it was determined that the inhibitory domain was manifest only in the mutant truncated to Trp-800 and was absent from that ending at Lys-793. These results indicate that a critical region of the inhibitory domain is contained within the sequence Tyr-794 to Trp-800. This region overlaps with the calmodulin-binding site for five residues. Our assignment of the inhibitory sequence is consistent with autoinhibition via a pseudosubstrate domain.
Ito
M
M
First Medical Clinic, Mie University Hospital, Mieken, Japan.
Guerriero
V
V
Chen
X M
XM
Hartshorne
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004789:Enzyme Activation / Q000187:drug effects; D005895:Gizzard, Avian / Q000201:enzymology; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology; D014422:Turkeys
D009219:Myosin-Light-Chain Kinase; D014357:Trypsin