Wu G, Wong A, Qian F, Lu R C
Muscle Research Group, Boston Biomedical Research Institute, Massachusetts 02114, USA.
Biochemistry. 1998 May 26;37(21):7676-85. doi: 10.1021/bi980054+.
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.
调节轻链（RLC）突变体RLC - C18和RLC - C165，分别在靠近N端和C端的第18和165位含有一个半胱氨酸，每个都用二苯甲酮4 - 碘乙酰胺标记，并以磷酸化或未磷酸化形式交换到肌球蛋白中，然后进行光解。SDS - PAGE显示，对于RLC - C18，肌球蛋白中的链内光交联被磷酸化抑制。对于含有RLC - C165的肌球蛋白，一条链内交联带的产量显著下降，而另一条不受磷酸化影响。结合质谱的肽图谱分析表明，Cys165与Ala17 - Lys34内的位点发生交联，与Ser19的磷酸化无关。这清楚地表明RLC的N端和C端区域之间的接近度不受磷酸化影响。此外，Cys165也可以与Phe133 - Arg143区域交联；然而，这种类型的交联在磷酸化状态下受到抑制。对于RLC - C18，交联仅在未磷酸化状态下与Glu124 - Arg132或Phe133 - Arg143区域发生。因此，磷酸化改变了Glu124 - Arg143区域与Cys18和Cys165之间的空间关系。在扇贝肌球蛋白中，对应于Glu124 - Arg143的区域位于RLC与必需轻链以及重链之间的界面处[谢，X.等人（1994年）《自然》368，306 - 312]。鉴于该研究，我们的结果表明Glu124 - Arg143区域参与磷酸化依赖性信号传导，并且其与RLC的N端和C端的空间关系变化可能是平滑肌肌球蛋白激活的基础。
