Phosphorylation changes the spatial relationship between Glu124-Arg143 and Cys18 and Cys165 of the regulatory light chain in smooth muscle myosin.
Regulatory light chain (RLC) mutants, RLC-C18 and RLC-C165, containing a single cysteine at positions 18 and 165 near the N and C terminus, respectively, were each labeled with benzophenone 4-iodoacetamide and exchanged into myosin in their phosphorylated or unphosphorylated forms and then photolyzed. SDS-PAGE showed that, for RLC-C18, the intrachain photo-cross-linking in myosin was inhibited by phosphorylation. For myosin containing RLC-C165, the yield of one intrachain cross-linked band decreased significantly whereas the other was unaffected by phosphorylation. Peptide mapping in conjunction with mass spectrometry showed that Cys165 was cross-linked to site(s) within Ala17-Lys34 independent of the phosphorylation of Ser19. This clearly demonstrates that the proximity between the N- and C-terminal regions of RLC is not affected by phosphorylation. In addition, Cys165 could also be cross-linked to the region of Phe133-Arg143; however, this type of cross-linking was inhibited in the phosphorylated state. For RLC-C18, the cross-linking took place with the region of Glu124-Arg132 or Phe133-Arg143, also only in the unphosphorylated state. Thus, phosphorylation changes the spatial relationship between the region of Glu124-Arg143 and Cys18 and Cys165. In scallop myosin, the region corresponding to Glu124-Arg143 is located at the interfaces between RLC and the essential light chain as well as the heavy chain [Xie, X. , et al. (1994) Nature 368, 306-312]. In light of that work, our results suggest that the region of Glu124-Arg143 is involved in the phosphorylation-dependent signaling and the change in its spatial relationship with respect to the N and C termini of RLC may underlie the activation of the smooth muscle myosin.
Wu
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Muscle Research Group, Boston Biomedical Research Institute, Massachusetts 02114, USA.
Wong
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Qian
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Lu
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D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002645:Chickens; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005895:Gizzard, Avian; D018698:Glutamic Acid / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008974:Mollusca; D009130:Muscle, Smooth / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D018994:Myosin Light Chains / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010777:Photochemistry
D003432:Cross-Linking Reagents; D018994:Myosin Light Chains; D018698:Glutamic Acid; D001120:Arginine; D003545:Cysteine