Structural and kinetic studies of phosphorylation-dependent regulation in smooth muscle myosin

Steven S. Rosenfeld, Jun Xing, Herbert C. Cheung, Fred Brown, Sambit Kar, H. Lee Sweeney

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Abstract

In this study, we have examined the mechanism of phosphorylation- dependent regulation in smooth muscle myosin through the use of structural and kinetic methodologies applied to several myosin fragments. Fluorescence anisotropy decay measurements demonstrate that regulatory light chain phosphorylation significantly reduces the rotational correlation time of regulatable myosin preparations, whereas minimally regulated ones show little effect in this assay. Sedimentation equilibrium studies show that the regulatory domain can dimerize with a dissociation constant that is unaffected by regulatory light chain phosphorylation. Finally, kinetic studies on the interactions of myosin-ADP constructs with actin are also consistent with a model in which interactions occur between the two heads, which are lost with regulatory light chain phosphorylation. We propose that in the absence of regulatory light chain phosphorylation, the two heads of myosin interact with each other, due to a weak intrinsic dimerization of the regulatory domains that is significantly stabilized by the proximal rod. Regulatory light chain phosphorylation abolishes the stabilizing effect of the proximal rod, leading to a loss of this interaction.

Original languageEnglish (US)
Pages (from-to)28682-28690
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number44
DOIs
StatePublished - Oct 30 1998

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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