PHOSPHORYLATION CHANGES THE SPATIAL RELATIONSHIP BETWEEN GLU124-ARG143 AND CYS18 AND CYS165 OF THE REGULATORY LIGHT-CHAIN IN SMOOTH-MUSCLE MYOSIN

Regulatory Light chain (ILC) 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 form s and then photolyzed. SDS-PAGE showed that, for RLC-C18, the intracha in 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 phosp horylation. Peptide mapping in conjunction with mass spectrometry show ed that Cys165 was cross-linked to site(s) within Ala17-Lys34 independ ent of the phosphorylation of Ser19. This clearly demonstrates that th e proximity between the N- and C-terminal regions of RLC is not affect ed by phosphorylation. In addition, Cys165 could also be cross-linked to the region of Phe133-Arg143; however, this type of cross-linking wa s inhibited in the phosphorylated state. For RLC-C18, the cross-linkin g took place with the region of Glu124-Arg132 or Phe133-Arg143, also o nly in the unphosphorylated state. Thus, phosphorylation changes the s patial relationship between the region of Glu124-Arg143 and Cys 18 and Cys 165. In scallop myosin, the region corresponding to Glu124-Arg143 is located at the interfaces between RLC and the essential light chai n as well as the heavy chain [Xie, X., et al. (1994) Nature 368, 306-3 12]. In light of that work, our results suggest that the region of Glu 124-Arg143 is involved in the phosphorylation-dependent signaling and the change in its spatial relationship with respect to the N and C ter mini of RLC may underlie the activation of the smooth muscle myosin.