Dynamics of myosin light chain phosphorylation at Ser¹⁹ and Thr¹⁸/Ser¹⁹in smooth muscle cells in culture

Abstract

Using the specific antibodies pLC1 and pLC2 for mono- and diphosphorylated 20-kDa myosin light chain (MLC20) at Ser19 and at both Thr18 and Ser19, respectively, we visualized the dynamics of the MLC20phosphorylation in rabbit aortic smooth muscle cells (cell line SM-3) stimulated with PGF2α. In the resting state, the diphosphorylated form was located in the peripheral region of the cell, such as the leading edge or the adhesion plaque, and the monophosphorylated form was located not only in the peripheral region but also on a discontinuous fibrillary structure along the long axis of the cell. After stimulation with 30 μM PGF2α, although localization of the monophosphorylated form changed little, the content of the diphosphorylated form increased and the distribution spread along the fibrillary structure to an extent the same as or similar to that of the monophosphorylated form, which colocalized with actin filament bundles. The diphosphorylation of MLC20 was more sensitive to protein kinase inhibitors, HA-1077, HA-1100, staurosporine, wortmannin, and ML-9, than was the monophosphorylation. In light of these observations, we propose that MLC20 diphosphorylation and monophosphorylation are regulated by different mechanisms.

Regulation of Vascular Smooth Muscle Tone by N-terminal Region of Caldesmon

“…For the comparison of LC 20 phosphorylation levels, cells were costained with a monoclonal (mouse) antibody (1:500) specific for phosphorylation of LC 20 at the MLCK sites, which was a gift from Drs. Y. Sasaki and M. Seto (Asahi Chemical Industry Co., Fuji, Japan) and has been previously characterized (31) and with rhodamine phalloidin (1: 2000, Molecular Probes) to label actin. For data analysis, a region was identified containing a filament bundle, and fluorescence for both LC 20 phosphorylation and actin were quantitated and integrated, and the ratio of LC 20 phosphorylation fluorescence/actin fluorescence was calculated.…”

Synergism of protein kinase A, protein kinase C, and myosin light-chain kinase in the secretory cascade of the pancreatic beta-cell.

“…The anti-phosphorylated MLC antibodies elucidated distinct mechanisms between mono-and diphosphorylation of rabbit smooth muscle MLC at the MLCK phosphorylatable sites (24). Application of these antibodies to immunostaining microscopy of the ␤-cell revealed that both mono-and diphosphorylated forms of MLC were distributed in the cell, even under nonstimulated conditions.…”

Differential effects of an expected actin-tropomyosin binding region of heat shock protein 20 on the relaxation in skinned carotid artery and taenia cecum from guinea pig

“…The dried preparations were then incubated in urea-sample buffer (containting 20 mM Trizma base, 10 mM DTT, 8M urea) for 8 hrs at 4°C. The extracts were subjected to glycerol-PAGE coupled with Western blot (Sakurada et al, 1998;Katayama et al, 2006). MLC20 was detected with anti-MLC20 antibody given by Dr. S. Yoshiyama of Gunma University and visualized with horseradish preoxidase-conjutated anti rabbit IgG (GE Healthcare UK, Buckinghamshire, UK) and enhanced chemiliminescence (ECL) Western blotting detection system (ECL+plus, GE Healthcare, UK).…”