Functional assembly of fragments from bisected smooth muscle myosin light chain kinase.
The C-terminal regulatory segment of smooth muscle myosin light chain kinase folds back on its catalytic core to inhibit kinase activity. This regulatory segment consists of autoinhibitory residues linking the catalytic core to the calmodulin-binding sequence and perhaps additional C-terminal residues including an immunoglobulin-like motif. However, mutational and biochemical analyses showed no specific involvement of residues C-terminal to the calmodulin-binding sequence. To obtain additional insights on the proposed mechanisms for autoinhibition and Ca(2+)/calmodulin activation of the kinase, the polypeptide backbone chain of myosin light chain kinase was cleaved by genetic means to produce N- and C-terminal protein fragments. The N-terminal fragment containing the catalytic core was catalytically inactive when expressed alone. Co-expression of the N-terminal fragment with the C-terminal fragment containing the regulatory segment restored kinase activity. Deletion of the autoinhibitory linker residues without or with the calmodulin-binding sequence prevented restoration of kinase activity. In the presence or absence of Ca(2+)/calmodulin, regulatory segment binding occurred through the linker region connecting the catalytic core to the calmodulin-binding sequence. Collectively, these results indicate that residues C-terminal to the calmodulin-binding sequence (including the immunoglobulin-like motif) are not functional components of the regulatory segment. Furthermore, the principal autoinhibitory motif is contained in the sequence linking the catalytic core of myosin light chain kinase to the calmodulin-binding sequence.
Padre
R C
RC
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9040, USA.
Stull
J T
JT
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D002645:Chickens; D017931:DNA Primers; D004789:Enzyme Activation; D004795:Enzyme Stability; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits
D002147:Calmodulin; D017931:DNA Primers; D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase; D002118:Calcium