Structural and kinetic studies of phosphorylation-dependent regulation in smooth muscle myosin.
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.
Rosenfeld
S S
SS
Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. s_rosenfeld@email.neuro.uab.edu
Xing
J
J
Cheung
H C
HC
Brown
F
F
Kar
S
S
Sweeney
H L
HL
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002645:Chickens; D004789:Enzyme Activation; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000737:chemistry; D010766:Phosphorylation; D011721:Pyrenes / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D062367:ortho-Aminobenzoates / Q000737:chemistry
D000199:Actins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D011721:Pyrenes; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; C048876:1,5-AEDANS; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; C030984:pyrene; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins