Salhia Bodour, Hwang Jeong Hyun, Smith Christian A, Nakada Mitsutoshi, Rutka Fiona, Symons Marc, Rutka James T
The Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, The University of Toronto, Toronto, Ontario, Canada.
Cell Motil Cytoskeleton. 2008 Jan;65(1):12-24. doi: 10.1002/cm.20240.
The generation of contractile force mediated by actin-myosin interactions is essential for cell motility. Myosin activity is promoted by phosphorylation of myosin light chain (MLC). MLC phosphorylation in large part is controlled by kinases that are effectors of Rho family GTPases. Accordingly, in this study we examined the effects of ROCK and Rac1 inhibition on MLC phosphorylation in astrocytoma cells. We found that low concentrations of the ROCK inhibitor Y27632 increased the phosphorylation state of the Triton X-100 soluble fraction of MLC, whereas higher concentrations of Y27632 decreased soluble phospho-MLC. These effects of Y27632 were dependent on Rac1. The soluble form of phospho-MLC comprises about 10% of total phospho-MLC in control cells. Interestingly, ROCK inhibition led to a decrease in the phosphorylation state of total MLC, whereas Rac1 inhibition had little effect. Thus, the soluble form of MLC is differentially regulated by ROCK and Rac1 compared with MLC examined in a total cell extract. We also observed that astrocytoma migration is stimulated by low concentrations of the myosin II inhibitor blebbistatin. However, higher concentrations of blebbistatin inhibit migration leading us to believe that migration has a biphasic dependence on myosin II activity. Taken together, our data show that modulation of myosin II activity is important in determining optimal astrocytoma migration. In addition, these findings suggest that there are at least two populations of MLC that are differentially regulated.
由肌动蛋白-肌球蛋白相互作用介导的收缩力产生对于细胞运动至关重要。肌球蛋白轻链（MLC）的磷酸化促进肌球蛋白活性。MLC磷酸化在很大程度上由作为Rho家族GTP酶效应器的激酶控制。因此，在本研究中，我们检测了ROCK和Rac1抑制对星形细胞瘤细胞中MLC磷酸化的影响。我们发现低浓度的ROCK抑制剂Y27632增加了MLC的Triton X-100可溶部分的磷酸化状态，而较高浓度的Y27632降低了可溶磷酸化MLC。Y27632的这些作用依赖于Rac1。在对照细胞中，磷酸化MLC的可溶形式约占总磷酸化MLC的10%。有趣的是，ROCK抑制导致总MLC的磷酸化状态降低，而Rac1抑制几乎没有影响。因此，与在全细胞提取物中检测的MLC相比，MLC的可溶形式受ROCK和Rac1的差异调节。我们还观察到低浓度的肌球蛋白II抑制剂blebbistatin刺激星形细胞瘤迁移。然而，较高浓度的blebbistatin抑制迁移，这使我们相信迁移对肌球蛋白II活性具有双相依赖性。综上所述，我们的数据表明，调节肌球蛋白II活性对于确定最佳的星形细胞瘤迁移很重要。此外，这些发现表明至少有两种不同调节的MLC群体。
