The human gastric cancer cell line HGC-27 was provided by the Institute of Cell Biology (Shanghai, China). Adenoviral vectors encoding the cDNA β-galactosidase (Ad-LacZ) and PKG II (Ad-PKG II) were provided by Dr Gerry Boss and Dr Renate Pilz (University of California, San Diego, CA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific Inc., Waltham, MA, USA). The polyclonal rabbit anti-human PKG II antibody was obtained from Abgent Biotechnology (San Diego, CA, USA; cat. no. AP8001a; dilution, 1:200). The horseradish peroxidase (HRP)-conjugated monoclonal mouse anti-human β-actin antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA; cat. no. sc-47778; dilution, 1:1,000). Polyclonal rabbit anti-p-ErbB2 (T686; cat. no. ab11717; dilution, 1:1,000) and polyclonal rabbit anti-human phosphoserine/threonine (cat. no. ab17464; dilution, 1:1,000) were purchased from Abcam (Cambridge, MA, USA). Polyclonal rabbit anti-human p-HER2 (Tyr1248) antibody (cat. no. BS4090; dilution, 1:500) and polyclonal rabbit anti-human ErbB2/HER2 (cat. no. BS1169; dilution, 1:500) were obtained from Bioworld Technology, Inc. (St. Louis Park, MN, USA). Monoclonal mouse anti-flag antibody (cat. no. F1804; dilution, 1:1,000) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The HRP-conjugated polyclonal anti-mouse and anti-goat IgG secondary antibodies (cat. nos. 115-035-003 and 111-035-003, respectively; dilution, 1:10,000) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The Bioepitope^® protein A+G Agarose IP was obtained from Bioworld Technology, Inc. The cellular permeable cGMP analog 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate (8-pCPT-cGMP) was acquired from Calbiochem (San Diego, CA, USA). EGF was purchased from Sigma-Aldrich. The cell transfection reagent Lipofectamine™ 2000 and E. coli BL-21DE3 were obtained from Invitrogen (Thermo Fisher Scientific, Inc.). The QuikChange Lightning Site-Directed Mutagenesis kit was purchased from Agilent Technologies (Santa Clara, CA, USA) and the SanPrep Column Plasmid Mini-Preps kit was obtained from Sangon Biotech Shanghai Co. Ltd. (Shanghai, China). Electrochemiluminescence (ECL) reagents were acquired from EMD Millipore (Billerica, MA, USA).

HGC-27 cells were cultured in DMEM supplemented with 10% FBS and maintained at 37°C in a humidified incubator with 95% air and 5% CO₂. On the day prior to infection, cells were planted into 6-well plates. To observe the phosphorylation of HER2, the cells were infected with Ad-LacZ or Ad-PKG II for 24 h and serum starved overnight. Subsequently, in the Ad-LacZ + EGF and Ad-PKG II + EGF groups, the cells were incubated with EGF (100 ng/ml) for 5 min; in the Ad-PKG II + cGMP + EGF groups, the cells were incubated with 8-pCPT-cGMP for 1 h and then with EGF (100 ng/ml) for 5 min. To observe PKGII binding with HER2 directly and causing the serine/threonine phosphorylation of HER2, the cells were infected with Ad-PKG II for 24 h, serum starved overnight and incubated with 8-pCPT-cGMP for 1 h. At the end of the treatments, the cells were harvested by aspiration of the media and direct addition of heated 2X SDS sample buffer. The cell lysate was scraped and transferred to tubes, heated for 5 min at 100°C and stored at −20°C.

The cells growing on the 100-mm culture plate were washed two times with cold PBS and lysed by adding 1 ml radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China; 50 mM Tris-HCl pH 7.4, 1% Triton X-100, 1 mM EDTA, 1 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 1 mM Na₃VO₄) per plate. An antibody against tag protein flag was used for immunoprecipitation. The precipitates were probed with antibodies against target proteins.

Proteins were separated by SDS-PAGE (10%) gel (EMD Millipore) according to the molecular size and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). Blots were blocked with 5% (w/v) non-fat milk in Tris-buffered saline with Tween 20 for 1 h at room temperature and then incubated at 4°C overnight with the primary antibodies (including anti-p-HER2, anti-β-actin, anti-p-ErbB2/HER2, anti-phosphoserine/threonine and anti-flag), followed by incubation with the secondary antibodies (including goat anti-mouse and goat anti-rabbit HRP-conjugated antibodies) at room temperature for 1 h. The signal was visualized using ECL detection reagents. To perform densitometry analysis, digital images of the positive bands were obtained with Chemidoc XRS and analyzed using the image analysis program Quantity One, version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The results are presented as the ratio of target protein/loading control.

The cDNA encoding human HER2 was cut off by HindIII from the plasmid CMV-HER2-WT (cat. no. 16257; Addgene, Cambridge, MA, USA) and was cloned into the expression vector p3XFlag-myc-CMV-24. Mutants of HER2 were generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene, San Diego, CA, USA). Threonine 686 was mutated to glutamic acid (T686E) and alanine (T686A). The following primers were used: Mutant HER2 (T686E), forward 5′-GAAGATCCGGAAGTACGAGATGCGGAGACTGCTG-3′ and reverse 5′-CAGCAGTCTCCGCATCTCGTACTTCCGGATCTTC-3′; mutant HER2 (T686A), forward 5′-GAAGATCCGGAAGTACGCGATGCGGAGACTGCTG-3′ and reverse CAGCAGTCTCCGCATCTCGTACTTCCGGATCTTC. The mutant plasmids were sequenced and the mutations were confirmed.

The data are expressed as the mean ± standard deviation. Statistical significance was performed using a two-tailed analysis of variance with SPSS statistical software, version 19 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The activation of HER2 is dependent on the ligand-receptor binding of other ErbB receptors, particularly EGFR. When EGF binds with EGFR, EGFR forms a dimer with HER2 and this dimerization causes autophosphorylation/activation of HER2 (5). Tyrosine 1248 (Tyr1248) is one of the autophosphorylation sites of HER2 and phosphorylation of this site is associated with downstream signaling (9). The present study investigated the inhibitory effect of PKG II on Tyr1248 phosphorylation of HER2 in differently treated HGC-27 cells using western blotting. The results demonstrated that in Ad-LacZ-infected cells, there was a pronounced increase in Tyr1248 phosphorylation of HER2 when the cells were incubated with EGF (100 ng/ml) for 5 min. In cells infected with Ad-PKG II for 24 h, treated with 8-pCPT-cGMP for 1 h and then incubated with EGF (100 ng/ml) for 5 min, the Tyr1248 phosphorylation of HER2 was significantly decreased (Fig. 1). This indicated that PKG II could inhibit EGF-induced Tyr1248 phosphorylation of HER2.

Since the phosphorylation/activation of HER2 was caused by EGF/EGFR binding and PKG II has been reported to bind with EGFR (10), whether PKG II directly inhibits HER2 or the inhibition is subsequent to its inhibition of EGFR requires further investigation. Co-IP was performed to detect the possible interaction between PKG II and HER2. The results demonstrated that in HGC-27 cells infected with Ad-PKG II and stimulated with 8-pCPT-cGMP, binding between PKG II and HER2 occurred (Fig. 2). Western blotting with antibodies against pan serine/threonine phosphorylation was used to detect the PKG II-induced serine/threonine phosphorylation of HER2. The results demonstrated that in cells infected with Ad-PKG II and treated with 8-pCPT-cGMP, there was a pronounced increase in serine/threonine phosphorylation of HER2 (Fig. 3). These results indicated that PKG II inhibited the tyrosine phosphorylation/activation of HER2 through directly binding with HER2 and causing phosphorylation.

In order to reveal the PKG II-specific phosphorylation site of HER2, western blotting with antibodies against p-HER2 (T686) was used to detect the phosphorylation of threonine 686 on HER2 in cells infected with Ad-PKG II and treated with 8-pCPT-cGMP. The results demonstrated that PKG II caused a pronounced increase in phosphorylation of threonine 686 (Fig. 4). To further determine if this site was the main site for PKG II-induced phosphorylation, plasmids encoding the cDNA of flag-tagged wild-type and mutant HER2 were constructed. The cells were transfected with the plasmids to express the mutants of HER2 and the proteins were isolated by immunoprecipitating with antibody against flag. Western blotting with antibody against pan serine/threonine phosphorylation was applied to detect the phosphorylation of the precipitated HER2 proteins. The results demonstrated that PKG II did not cause serine/threonine phosphorylation of the T686A mutant of HER2 and had no inhibitory effect on HER2 tyrosine phosphorylation/activation, confirming that threonine 686 was the main phosphorylating site of PKG II. Furthermore, EGF had no stimulating effect on the T686E mutant of HER2, indicating that the mimic of threonine 686 phosphorylation prevented the activation of HER2 by EGF (Fig. 5). These results confirmed that threonine 686 was the PKG II-specific phosphorylation site of HER2 and the phosphorylation of this site was crucial for the inhibition of HER2 activation by PKG II.