Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40)
Date
2002
Authors
Van Lierop, J.
Wilson, D.
Davis, J.
Tikunova, S.
Sutherland, C.
Walsh, M.
Johnson, J.
Editors
Advisors
Journal Title
Journal ISSN
Volume Title
Type:
Journal article
Citation
Journal of Biological Chemistry, 2002; 277(8):6550-6558
Statement of Responsibility
Jacquelyn E. Van Lierop, David P. Wilson, Jonathan P. Davis, Svetlana Tikunova, Cindy Sutherland, Michael P. Walsh, and J. David Johnson
Conference Name
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.
School/Discipline
Dissertation Note
Provenance
Published online ahead of print December 17, 2001
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Rights
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.