Our previous study found that Type II cyclic guanosine monophosphate-dependent protein kinase (PKG II) inhibited VEGF-induced tyrosine phosphorylation/ activation of VEGFR-2. But how does PKG II inhibit VEGFR-2 is not clear yet. The aim of this paper was to investigate the molecular mechanism of PKG II inhibition on VEGFR-2. Human gastric cancer cell line HGC-27 and Human Umbilical Vein Endothelial Cells (HUVECs) were infected with adenoviral construct encoding cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II and treated with 8-pCPT-cGMP to activate the kinase. Trans-well migration assay results showed that PKG II inhibited VEGF-induced migration of gastric cancer cells. Tube formation assay results showed that PKG II inhibited VEGF-induced tube formation of HUVEC cells. Co- Immunoprecipitation results indicated that PKG II combined with VEGFR-2. Immunoprecipitation and Western blotting results showed that PKG II caused Serine/Threonine phosphorylation of VEGFR-2. Mutagenesis and Western blotting results showed that when Threonine 439 and Serine1231 of VEGFR-2 were mutated to Alanine which could not be phosphorylated, the inhibition of PKG II on VEGFR-2 disappeared. The results suggest that PKG II inhibits VEGF-induced activation of VEGFR-2, migration and tube formation through phosphorylating Threonine439 and Serine1231 of VEGFR-2.
Ying Wang, Weihui Zhang, Yan Wu, Min Wu, Hai Qian, Yongchang Chen
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