Ramachandran C, Patil R V, Combrink K, Sharif N A, Srinivas S P
School of Optometry, Indiana University, Bloomington, IN 47405, USA.
Mol Vis. 2011;17:1877-90. Epub 2011 Jul 14.
The outflow facility for aqueous humor across the trabecular meshwork (TM) is enhanced by agents that oppose the actomyosin contraction of its resident cells. Phosphorylation of MYPT1 (myosin light chain [MLC] phosphatase complex of Type 1) at Thr853 and Thr696 inhibits dephosphorylation of MLC, leading to an increase in actomyosin contraction. In this study, we examined the effects of Rho kinase (ROCK) inhibitors on the relative dephosphorylation of the two sites of MYPT1 using human TM cells (GTM3).
Dephosphorylation of MYPT1 at Thr853 and Thr696 was determined by western blot analysis following exposure to selective inhibitors of ROCK, namely Y-27632 and Y-39983. Consequent dephosphorylation of MLC and decreases in actomyosin contraction were assessed by western blot analysis and collagen gel contraction assay, respectively. Changes in the cell-matrix adhesion were measured in real time by electric cell-substrate impedance sensing and also assessed by staining for paxillin, vinculin, and focal adhesion kinase (FAK).
Both ROCK inhibitors produced a concentration-dependent dephosphorylation at Thr853 and Thr696 of MYPT1 in adherent GTM3 cells. IC₅₀ values for Y-39983 were 15 nM and 177 nM for dephosphorylation at Thr853 and Thr696, respectively. Corresponding values for Y-27632 were 658 nM and 2270 nM. Analysis of the same samples showed a decrease in MLC phosphorylation with IC₅₀ values of 14 nM and 1065 nM for Y-39983 and Y-27632, respectively. Consistent with these changes, both inhibitors opposed contraction of collagen gels induced by TM cells. Exposure of cells to the inhibitors led to a decrease in the electrical cell-substrate resistance, with the effect of Y-39983 being more pronounced than Y-27632. Treatment with these ROCK inhibitors also showed a loss of stress fibers and a concomitant decrease in tyrosine phosphorylation of paxillin and FAK.
Y-39983 and Y-27632 oppose ROCK-dependent phosphorylation of MYPT1 predominantly at Thr853 with a corresponding decrease in MLC phosphorylation. A relatively low effect of both ROCK inhibitors at Thr696 suggests a role for other Ser/Thr kinases at this site. Y-39983 was several-fold more potent when compared with Y-27632 at inhibiting the phosphorylation of MYPT1 at either Thr853 or Thr696 commensurate with its greater potency at inhibiting the activity of human ROCK-I and ROCK-II enzymes.
通过抑制小梁网（TM）驻留细胞的肌动球蛋白收缩的药物可增强房水经小梁网的流出功能。MYPT1（1型肌球蛋白轻链[MLC]磷酸酶复合物）在苏氨酸853和苏氨酸696位点的磷酸化会抑制MLC的去磷酸化，导致肌动球蛋白收缩增加。在本研究中，我们使用人小梁网细胞（GTM3）研究了Rho激酶（ROCK）抑制剂对MYPT1两个位点相对去磷酸化的影响。
在暴露于ROCK的选择性抑制剂（即Y-27632和Y-39983）后，通过蛋白质印迹分析测定MYPT1在苏氨酸853和苏氨酸696位点的去磷酸化。分别通过蛋白质印迹分析和胶原凝胶收缩试验评估随后的MLC去磷酸化和肌动球蛋白收缩的降低。通过电细胞基质阻抗传感实时测量细胞-基质粘附的变化，并通过桩蛋白、纽蛋白和粘着斑激酶（FAK）染色进行评估。
两种ROCK抑制剂在贴壁的GTM3细胞中均在MYPT1的苏氨酸853和苏氨酸696位点产生浓度依赖性去磷酸化。Y-39983在苏氨酸853和苏氨酸696位点去磷酸化的IC₅₀值分别为15 nM和177 nM。Y-27632的相应值为658 nM和2270 nM。对相同样品的分析显示MLC磷酸化降低，Y-39983和Y-27632的IC₅₀值分别为14 nM和1065 nM。与这些变化一致，两种抑制剂均抑制TM细胞诱导的胶原凝胶收缩。细胞暴露于抑制剂导致细胞-基质电阻降低，Y-39983的作用比Y-27632更明显。用这些ROCK抑制剂处理还显示应力纤维丧失，同时桩蛋白和FAK的酪氨酸磷酸化降低。
Y-39983和Y-27632主要在苏氨酸8S3位点对抗ROCK依赖性的MYPT1磷酸化，同时MLC磷酸化相应降低。两种ROCK抑制剂在苏氨酸696位点的作用相对较低，表明该位点存在其他丝氨酸/苏氨酸激酶的作用。与Y-27632相比，Y-39983在抑制MYPT1在苏氨酸853或苏氨酸696位点的磷酸化方面效力高几倍，与其在抑制人ROCK-I和ROCK-II酶活性方面更强的效力相称。
Zotero 插件
