Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40).
Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction. A soybean isoform of CaM, SCaM-4 (77% identical to human CaM) fails to activate MLCK, whereas SCaM-1 (90.5% identical to human CaM) is as effective as CaM. We exploited this difference to gain insights into the structural requirements in CaM for activation of MLCK. A chimera (domain I of SCaM-4 and domains II-IV of SCaM-1) behaved like SCaM4, and analysis of site-specific mutants of SCaM-1 indicated that K30E and G40D mutations were responsible for the reduction in activation of MLCK. Competition experiments showed that SCaM-4 binds to the CaM-binding site of MLCK with high affinity. Replacement of CaM in skinned smooth muscle by exogenous CaM or SCaM-1, but not SCaM-4, restored Ca(2+)-dependent contraction. K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca(2+)-induced contraction to CaM-depleted skinned smooth muscle, consistent with their capacity to activate MLCK. Interpretation of these results in light of the high-resolution structures of (Ca(2+))(4)-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys(30) and Gly(40) and residues from the C-terminal domain is created upon binding to MLCK, formation of which is required for activation of MLCK. Interactions between this activation domain and a region of MLCK distinct from the known CaM-binding domain are required for removal of the autoinhibitory domain from the active site, i.e., activation of MLCK, or this domain may be required to stabilize the conformation of (Ca(2+))(4)-CaM necessary for MLCK activation.
Van Lierop
Jacquelyn E
JE
Smooth Muscle Research Group and the Canadian Institutes of Health Research Group in Regulation of Vascular Contractility, Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada.
Wilson
David P
DP
Davis
Jonathan P
JP
Tikunova
Svetlana
S
Sutherland
Cindy
C
Walsh
Michael P
MP
Johnson
J David
JD
D000595:Amino Acid Sequence; D001622:Betaine; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000378:metabolism
D002147:Calmodulin; D020033:Protein Isoforms; D011994:Recombinant Proteins; D001622:Betaine; D009219:Myosin-Light-Chain Kinase; D008239:Lysine