Sakurada K, Seto M, Sasaki Y
Frontier 21 Project, Life Science Research Center, Asahi Chemical Industry Company, Ltd., Samejima, Fuji, Shizuoka 416-0934, Japan.
Am J Physiol. 1998 Jun;274(6):C1563-72. doi: 10.1152/ajpcell.1998.274.6.C1563.
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 MLC20 phosphorylation in rabbit aortic smooth muscle cells (cell line SM-3) stimulated with PGF2alpha. 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 microM PGF2alpha, 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.
分别使用针对20 kDa肌球蛋白轻链（MLC20）在Ser19处单磷酸化以及在Thr18和Ser19两处双磷酸化的特异性抗体pLC1和pLC2，我们观察了用前列腺素F2α（PGF2α）刺激的兔主动脉平滑肌细胞（细胞系SM-3）中MLC20磷酸化的动态变化。在静息状态下，双磷酸化形式位于细胞的周边区域，如前沿或黏附斑，而单磷酸化形式不仅位于周边区域，还位于沿细胞长轴的不连续纤维状结构上。在用30 microM PGF2α刺激后，尽管单磷酸化形式的定位变化不大，但双磷酸化形式的含量增加，其分布沿纤维状结构扩展至与单磷酸化形式相同或相似的程度，且与肌动蛋白丝束共定位。MLC20的双磷酸化比单磷酸化对蛋白激酶抑制剂HA-1077、HA-1100、星形孢菌素、渥曼青霉素和ML-9更为敏感。基于这些观察结果，我们提出MLC20的双磷酸化和单磷酸化受不同机制调控。
