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Type: Journal article
Title: Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40)
Author: Van Lierop, J.
Wilson, D.
Davis, J.
Tikunova, S.
Sutherland, C.
Walsh, M.
Johnson, J.
Citation: Journal of Biological Chemistry, 2002; 277(8):6550-6558
Publisher: Amer Soc Biochemistry Molecular Biology Inc
Issue Date: 2002
ISSN: 0021-9258
Statement of
Jacquelyn E. Van Lierop, David P. Wilson, Jonathan P. Davis, Svetlana Tikunova, Cindy Sutherland, Michael P. Walsh, and J. David Johnson
Abstract: 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, wheras 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 Ca2+-dependent contraction. K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca2+-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 (Ca2+)4-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys30 and Gly40 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 (Ca2+)4-CaM necessary for MLCK activation.
Keywords: Humans
Escherichia coli
Myosin-Light-Chain Kinase
Protein Isoforms
Recombinant Proteins
Cloning, Molecular
Sequence Alignment
Binding Sites
Enzyme Activation
Amino Acid Sequence
Protein Conformation
Sequence Homology, Amino Acid
Models, Molecular
Molecular Sequence Data
Provenance: Published online ahead of print December 17, 2001
Rights: © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
DOI: 10.1074/jbc.M111404200
Appears in Collections:Aurora harvest 6
Molecular and Biomedical Science publications

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