Department of Physiology, School of Medicine, Jiangsu University, Zhenjiang City, Jiangsu Province, China; School of Medical Technology, Xuzhou Medical University, Xuzhou City, Jiangsu Province, China.
Department of Physiology, School of Medicine, Jiangsu University, Zhenjiang City, Jiangsu Province, China.
Biochem Biophys Res Commun. 2019 Oct 8;518(1):14-18. doi: 10.1016/j.bbrc.2019.07.126. Epub 2019 Aug 6.
Our previous study demonstrated that type II cGMP-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF) induced tyrosine phosphorylation/activation of the EGF receptor (EGFR). This paper was designed to investigate the mechanism of the inhibition of PKG II on EGFR activation. Gastric cancer cells HGC-27 and AGS were infected with an adenoviral vector encoding the cDNA of PKG II (Ad-PKG II) to overexpress PKG II and treated with 8-(4-chlorophenylthio) guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) to activate the kinase. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assay were performed to detect the interaction between PKG II and EGFR. Western blotting, mass spectrometry (MS) and site mutagenesis were performed to detect the PKG II-specific phosphorylation site on EGFR. The results showed that in living COS-7 cells, which were infected with Ad-PKG II and treated with 8-pCPT-cGMP, there was an interaction between PKG II and EGFR. The results also showed that PKG II caused threonine 669 (T669) phosphorylation of EGFR in HGC-27 and AGS cells infected with Ad-PKG II and treated with 8-pCPT-cGMP, and then inhibited the activation of EGFR. When T669 of EGFR was mutated to alanine, the inhibitory effect of PKG II on the activation of EGFR was eradicated. These findings suggested a PKG II-specific phosphorylation site on EGFR, and might be beneficial to illuminate the anti-tumor role of PKG II.
我们之前的研究表明，II 型环鸟苷酸依赖性蛋白激酶（PKG II）抑制表皮生长因子（EGF）诱导的 EGF 受体（EGFR）酪氨酸磷酸化/激活。本文旨在研究 PKG II 抑制 EGFR 激活的机制。用编码 PKG II cDNA 的腺病毒载体（Ad-PKG II）感染胃癌细胞 HGC-27 和 AGS，以过表达 PKG II，并用 8-（4-氯苯硫基）鸟苷-3'，5'-环单磷酸（8-pCPT-cGMP）激活激酶。进行共免疫沉淀（Co-IP）和双分子荧光互补（BiFC）测定以检测 PKG II 和 EGFR 之间的相互作用。进行 Western blot、质谱（MS）和位点突变检测 EGFR 上 PKG II 特异性磷酸化位点。结果表明，在感染 Ad-PKG II 并用 8-pCPT-cGMP 处理的活 COS-7 细胞中，PKG II 和 EGFR 之间存在相互作用。结果还表明，PKG II 导致感染 Ad-PKG II 并用 8-pCPT-cGMP 处理的 HGC-27 和 AGS 细胞中 EGFR 的苏氨酸 669（T669）磷酸化，然后抑制 EGFR 的激活。当 EGFR 的 T669 突变为丙氨酸时，PKG II 对 EGFR 激活的抑制作用被消除。这些发现表明 EGFR 上存在 PKG II 特异性磷酸化位点，这可能有助于阐明 PKG II 的抗肿瘤作用。
