Consequently, EGFR is the initial growth factor receptor taken as cancer treatment target. Quite a few procedures of inhibiting EGFR action and related signal transduc tion, including distinct antibodies against EGFR and inhibitors of EGFR, received intensive analysis. New and much more useful techniques in blocking EGFR mediated signal transduction shall be valuable in cancer therapy. Thus, the acquiring that PKG II can inhibit the activation of EGFR and also the consequent signal transduction has vital significance. It strongly suggests that PKG II is really a probable endogenous EGFR inhibitor and can give new hint on system of cancer therapy. Research data showed that some protein kinases may cause phosphorylation of EGFR and have an impact on its activation and or destination. One example is, protein kinase C may cause the phosphorylation of Threonine 654 of EGFR and regulates receptor binding and internalization.
Serine 1046 1047 phosphorylation internet site is needed for EGFR desensitization in selleck EGF treated cells. In our experiments, we detected the phosphorylation of Thr654 and Ser1046 1047 of EGFR and identified that PKG II didn’t result in the phosphorylation of those phosphorylation sites. However, our benefits showed that there was a direct interaction amongst PKG II and EGFR and PKG II brought on Threonine phosphorylation of EGFR. This indicated that PKG II blocked the activation of EGFR by way of binding with and phosphorylating the receptor. The precise phosphorylation webpage might be our subsequent analysis aim. as well as the activation of PKC and CAMK IIa. To the inhibition of Products and Methods Cell Line and Reagents Human gastric cancer cell line AGS was supplied through the Institute of Cell Biology. Adenoviral vectors encoding b galactosidase and PKG II were kind gifts from Dr. Gerry Boss and Dr.
Renate Pilz in selleck chemical University of California, San Diego, U. S. A. Dulbeccos Modified Eagles Media and fetal bovine serum had been from GIBCO. The antibody towards PKG II was from ABGENT Biotechnology. Goat anti b actin, mouse anti pan Ras, and mouse anti PLCc1 antibodies had been from Santa Cruz. Rabbit anti p EGFR, rabbit anti p EGFR, rabbit anti EGFR, mouse anti p ERK, rabbit anti ERK and rabbit anti p PLCc1 antibodies have been from Cell Signaling Technology. Rabbit anti PKCa and rabbit anti p CaMK IIa have been from Bioworld Engineering. Rabbit anti p Thr antibody was from Abcam. Horseradish peroxidase conjugated secondary anti bodies had been from Jackson Immuno Analysis Laboratories. The cellular permeable cGMP analog eight pCPT cGMP was from Calbiochem. EGF, U73122 and U0126 were from Sigma. Electrochemiluminescence reagents had been from Millipore. Ca2 indicator Fluo three AM and Membrane and Cytosol Protein Extraction Kit had been from Beyotime.