Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40)

J Biol Chem. 2002 Feb 22;277(8):6550-8. doi: 10.1074/jbc.M111404200. Epub 2001 Dec 17.

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, 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Betaine*
  • Binding Sites
  • Calmodulin / metabolism
  • Calmodulin / pharmacology*
  • Cloning, Molecular
  • Enzyme Activation
  • Escherichia coli
  • Humans
  • Kinetics
  • Lysine*
  • Models, Molecular
  • Molecular Sequence Data
  • Myosin-Light-Chain Kinase / chemistry
  • Myosin-Light-Chain Kinase / genetics
  • Myosin-Light-Chain Kinase / metabolism*
  • Protein Conformation
  • Protein Isoforms / chemistry
  • Protein Isoforms / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Soybeans / metabolism

Substances

  • Calmodulin
  • Protein Isoforms
  • Recombinant Proteins
  • Betaine
  • Myosin-Light-Chain Kinase
  • Lysine