Consequently, EGFR may be the to begin with growth element receptor taken as cancer treatment target. Numerous tactics of inhibiting EGFR activity and related signal transduc tion, including particular antibodies towards EGFR and inhibitors of EGFR, acquired intensive investigate. New and much more helpful solutions in blocking EGFR mediated signal transduction will be helpful in cancer therapy. Therefore, the obtaining that PKG II can inhibit the activation of EGFR plus the consequent signal transduction has significant significance. It strongly suggests that PKG II is actually a potential endogenous EGFR inhibitor and will give new hint on strategy of cancer therapy. Analysis data showed that some protein kinases could cause phosphorylation of EGFR and impact its activation and or location. One example is, protein kinase C can cause the phosphorylation of Threonine 654 of EGFR and regulates receptor binding and internalization.
Serine 1046 1047 phosphorylation web site is needed for EGFR desensitization in a replacement EGF treated cells. In our experiments, we detected the phosphorylation of Thr654 and Ser1046 1047 of EGFR and located that PKG II did not lead to the phosphorylation of those phosphorylation internet sites. Yet, our outcomes showed that there was a direct interaction between PKG II and EGFR and PKG II triggered Threonine phosphorylation of EGFR. This indicated that PKG II blocked the activation of EGFR through binding with and phosphorylating the receptor. The precise phosphorylation website is going to be our up coming analysis target. and the activation of PKC and CAMK IIa. For your inhibition of Components and Approaches Cell Line and Reagents Human gastric cancer cell line AGS was offered by the Institute of Cell Biology. Adenoviral vectors encoding b galactosidase and PKG II were sort presents from Dr. Gerry Boss and Dr.
Renate Pilz in selleck chemicals compound libraries University of California, San Diego, U. S. A. Dulbeccos Modified Eagles Media and fetal bovine serum were from GIBCO. The antibody against PKG II was from ABGENT Biotechnology. Goat anti b actin, mouse anti pan Ras, and mouse anti PLCc1 antibodies have 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 Engineering. Rabbit anti PKCa and rabbit anti p CaMK IIa were from Bioworld Technology. Rabbit anti p Thr antibody was from Abcam. Horseradish peroxidase conjugated secondary anti bodies were from Jackson Immuno Study Laboratories. The cellular permeable cGMP analog 8 pCPT cGMP was from Calbiochem. EGF, U73122 and U0126 have been from Sigma. Electrochemiluminescence reagents were from Millipore. Ca2 indicator Fluo 3 AM and Membrane and Cytosol Protein Extraction Kit had been from Beyotime.