Proteolytic cleavage sites in smooth muscle myosin-light-chain kinase and their relation to structural and regulatory domains.
Proteolysis of the smooth muscle myosin-light-chain kinase with either thermolysin or endoproteinase Lys-C cleaves the enzyme towards the amino-terminus between the first and second unc domains, unc-II-1 and unc-II-2, and in the calmodulin-binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys-C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys-C endoproteinase results in a constitutively active 474-residue fragment with the same amino-terminus, but a carboxyl-terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic-residue-rich connecting peptide between the amino-terminus of the catalytic domain and the unc-I domain, nor within the unc-II or unc-I domains or between the adjacent unc-II-2 and unc-I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin-light-chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK(787-807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777-793) proposed by Ikebe et al. (1989) [Ikebe, M., Maruta, S. & Reardon, S. (1989) J. Biol. Chem. 264, 6967-6971] to be important in autoregulation of the myosin-light-chain kinase.
St. Vincent's Institute of Medical Research, Fitzroy, Australia.
R B
RB
Pearson
M
M
Ito
N A
NA
Morrice
A J
AJ
Smith
R
R
Condron
R E
RE
Wettenhall
B E
BE
Kemp
D J
DJ
Hartshorne
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D003488:Cyanogen Bromide; D010450:Endopeptidases / Q000378:metabolism*; D005895:Gizzard, Avian / Q000201:enzymology; D007700:Kinetics; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014422:Turkeys
D002147:Calmodulin; D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase; D010450:Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D013820:Thermolysin; D003488:Cyanogen Bromide