Smooth muscle myosin filament assembly under control of a kinase-related protein (KRP) and caldesmon |
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Authors: | Kudryashov Dmitry S Vorotnikov Alexander V Dudnakova Tatyana V Stepanova Olga V Lukas Thomas J Sellers James R Watterson D Martin Shirinsky Vladimir P |
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Affiliation: | (1) Laboratory of Cell Motility, Cardiology Research Center, 3rd Cherepkovskaya street 15A, Moscow, 121552, Russia;(2) Present address: Department of Chemistry and Biochemistry, ULCA, Los Angeles, CA 90095, USA;(3) Laboratory of Molecular Cardiology, NHLBI NIH, Bethesda, MD 20892, USA |
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Abstract: | Kinase-related protein (KRP) and caldesmon are abundant myosin-binding proteins of smooth muscle. KRP induces the assembly
of unphosphorylated smooth muscle myosin filaments in the presence of ATP by promoting the unfolded state of myosin. Based
upon electron microscopy data, it was suggested that caldesmon also possessed a KRP-like activity (Katayama et al., 1995, J Biol Chem 270: 3919–3925). However, the nature of its activity remains obscure since caldesmon does not affect the equilibrium between
the folded and unfolded state of myosin. Therefore, to gain some insight into this problem we compared the effects of KRP
and caldesmon, separately, and together on myosin filaments using turbidity measurements, protein sedimentation and electron
microscopy. Turbidity assays demonstrated that KRP reduced myosin filament aggregation, while caldesmon had no effect. Additionally,
neither caldesmon nor its N-terminal myosin binding domain (N152) induced myosin polymerization at subthreshold Mg2+ concentrations in the presence of ATP, whereas the filament promoting action of KRP was enhanced by Mg2+. Moreover, the amino-terminal myosin binding fragment of caldesmon, like the whole protein, antagonizes Mg2+-induced myosin filament formation. In electron microscopy experiments, caldesmon shortened myosin filaments in the presence
of Mg2+ and KRP, but N152 failed to change their appearance from control. Therefore, the primary distinction between caldesmon and
KRP appears to be that caldesmon interacts with myosin to limit filament extension, while KRP induces filament propagation
into defined polymers. Transfection of tagged-KRP into fibroblasts and overlay of fibroblast cytoskeletons with Cy3KRP demonstrated
that KRP colocalizes with myosin structures in vivo. We propose a new model that through their independent binding to myosin and differential effects on myosin dynamics, caldesmon
and KRP can, in concert, control the length and polymerization state of myosin filaments.
This revised version was published online in July 2006 with corrections to the Cover Date. |
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