title: Type II cGMP‑dependent protein kinase inhibits RhoA activation in gastric cancer cells.
abstract: 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.
authors.affiliation: Department of Physiology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Physiology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China.
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Physiology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China.
authors.forename: Yueying
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Physiology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China.
authors.forename: Ting
authors.initials: T
authors.lastname: Lan
authors.affiliation: Department of Physiology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China.
authors.forename: Hai
authors.initials: H
authors.lastname: Qian
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002522:Chlorocebus aethiops; D062967:Cyclic GMP-Dependent Protein Kinase Type II / Q000737:chemistry / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013274:Stomach Neoplasms / Q000201:enzymology* / Q000473:pathology; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000199:Actins; D008246:Lysophospholipids; D010768:Phosphoserine; D062967:Cyclic GMP-Dependent Protein Kinase Type II; C569729:PRKG2 protein, human; D020742:rhoA GTP-Binding Protein; C032881:lysophosphatidic acid