Wang Ying, Chen Yongchang, Li Yueying, Lan Ting, Qian Hai
Department of Physiology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China.
Mol Med Rep. 2014 Apr;9(4):1444-52. doi: 10.3892/mmr.2014.1960. Epub 2014 Feb 18.
Small GTPase RhoA is a key signaling component regulating cell migration and stress fiber formation. Previous studies have shown that RhoA activity is regulated by protein kinases, such as cAMP‑dependent protein kinase (PKA) and type I cGMP‑dependent protein kinase (PKGI), which phosphorylate the protein. This study was designed to investigate the effect of type II cGMP‑dependent protein kinase (PKGII) on RhoA activity. Cells of the human gastric cancer line AGS were infected with adenoviral constructs bearing the PKGII cDNA in order to increase its endogenous expression, and were treated with 8‑pCPT‑cGMP to activate the PKGII enzyme. A transwell assay was performed to measure the migratory activity of the treated cells, and immunofluorescent microscopy was used to observe the formation of stress fibers. The phosphorylation of RhoA was detected by western blotting, and the activity of RhoA was measured by a pull‑down assay. Co‑immunoprecipitation (co‑IP) was performed to detect binding of PKGII to RhoA. Glutathione S‑transferase (GST)‑fused fragments of RhoA and PKGII were expressed in Escherichia coli and used to investigate the domains required for the binding. The results showed that lysophosphatidic acid (LPA) treatment increased the migration and the formation of stress fibers in AGS cells and that this effect was RhoA‑dependent. An increase in PKGII activity, not only inhibited LPA‑induced migration and stress fiber formation, but also suppressed LPA‑induced activation of RhoA. PKGII caused serine 188 (Ser188) phosphorylation of RhoA, but not the phosphorylation of the mutant RhoA Ser188A, and therefore had no inhibitory effect on the activity of the mutant protein. Co‑IP results showed that there is direct binding of PKGII to RhoA. The GST pull‑down assay showed that the fragment containing RhoA amino acid residues 1‑44 and the N‑terminal fragment of PKGII containing amino acid residues 1‑176 are required for the binding between the two proteins. These results suggested that PKGII inhibits RhoA activity by binding to this small GTPase and causing phosphorylation at its Ser188 site.
小GTP酶RhoA是调节细胞迁移和应力纤维形成的关键信号成分。先前的研究表明，RhoA活性受蛋白激酶调节，如环磷酸腺苷依赖性蛋白激酶（PKA）和I型环磷酸鸟苷依赖性蛋白激酶（PKGI），它们可使该蛋白磷酸化。本研究旨在探讨II型环磷酸鸟苷依赖性蛋白激酶（PKGII）对RhoA活性的影响。用人胃癌细胞系AGS感染携带PKGII cDNA的腺病毒构建体以增加其内源性表达，并用8-pCPT-cGMP处理以激活PKGII酶。进行Transwell试验以测量处理后细胞的迁移活性，并用免疫荧光显微镜观察应力纤维的形成。通过蛋白质印迹法检测RhoA的磷酸化，并通过下拉试验测量RhoA的活性。进行免疫共沉淀（co-IP）以检测PKGII与RhoA的结合。RhoA和PKGII的谷胱甘肽S-转移酶（GST）融合片段在大肠杆菌中表达，并用于研究结合所需的结构域。结果表明，溶血磷脂酸（LPA）处理增加了AGS细胞的迁移和应力纤维的形成，且这种作用依赖于RhoA。PKGII活性的增加不仅抑制了LPA诱导的迁移和应力纤维形成，还抑制了LPA诱导的RhoA激活。PKGII导致RhoA的丝氨酸188（Ser188）磷酸化，但不导致突变型RhoA Ser188A的磷酸化，因此对突变蛋白的活性没有抑制作用。免疫共沉淀结果表明PKGII与RhoA存在直接结合。GST下拉试验表明，RhoA的含氨基酸残基1-44的片段和PKGII的含氨基酸残基1-176的N端片段是两种蛋白之间结合所必需的。这些结果表明，PKGII通过与这种小GTP酶结合并在其Ser188位点引起磷酸化来抑制RhoA活性。
