Structural and Kinetic Studies of Phosphorylation-dependent Regulation in Smooth Muscle Myosin*
- From the Departments of ‡Neurology and¶Biochemistry and Molecular Genetics, and the **Graduate Program in Biophysical Sciences, University of Alabama at Birmingham, Birmingham, Alabama 35294 and the ‖Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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.
Footnotes
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↵* This work was supported by National Institutes of Health Grants NS 34856 and NS 31096 from the NINDS (to S. S. R.), by Grant AR 31239 from the NIAMSD (to H. C. C.), and by Grant AR35661 (to H. L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Dept. of Neurology, University of Alabama at Birmingham, UAB Station, Birmingham, AL 35294. Tel.: 205-934-1436; Fax: 205-975-7546; E-mail: s_rosenfeld{at}email.neuro.uab.edu.
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↵2 H. L. Sweeney, unpublished data.
- Abbreviations:
- RLC
-
myosin regulatory light chain
- ELC
-
myosin essential light chain
- HMM
-
heavy meromyosin
- S-1
-
myosin subfragment 1
- S-2
-
myosin subfragment 2
- mant ADP
-
N-methylanthraniloyl-ADP
- 1
-
5-IAEDANS, 5-[[[2-(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid
- DTT
-
dithiothreitol
- BeF
-
beryllium fluoride.
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- Received March 4, 1998.
- Revision received July 28, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
