Effect Of PKG Ⅱ On EGF/EGFR-induced Protein Expression And Protein Phosphorylation In Gastric Cancer Cells

PKG Ⅱ is a serine/threonine protein kinase which regulates cell signal transduction through phosphorylating specific serine / threonine sites.Our previous study found that PKG Ⅱ is a potential EGFR inhibitor by phosphorylating EGFR,inhibitingits activation and its downstream signaling pathways,thereby affecting biological activity such as tumor cell proliferation,migration and invasion.EGFR is a tyrosine kinase receptor which is overexpressed in many tumor types.EGFR overexpression,activation or mutation promotes tumor proliferationand metastasis.Activation of the kinase domain of EGFR increases the kinase activity of EGFR,leading to overactivation of downstream signaling and multiple cellular activities.During these changes,protein expression and protein phosphorylation are included.Our previous research has systemically elucidated the inhibition of PKG Ⅱ on EGF/EGFR mediated signaling and cellular activities.In order to further understand the inhibition of PKG Ⅱ on EGFR,this study investigated the effect of PKG Ⅱ on EGFR activation mediated protein expression and protein phosphorylation by applying two-dimensional electrophoresis and mass spectrometry.For the protein expression,we firstly investigated the inhibition of PKG Ⅱ on the known EGFR mediated protein expressions.Through data researching,wefound that EGF treatment could change the expression level of 161 proteins.Among them,the expression of 102 proteins was increased and the expression of 49 proteins was decreased by treatment with EGF.We selected three proteins,MMP1(cell migration),c-MYC(cell proliferation)and NOS1(NO synthase),as the observation objects and detected their expression by Western blotting.The results showed that in gastric cancer cell lines AGS and HGC-27,PKG Ⅱ was able to efficiently inhibit the increase of the expression of MMP-1,c-MYC and NOS1 proteins induced by EGF treatment.Further,we identified the expression 17 new proteins in AGS cell line by two-dimensional electrophoresis and mass spectrometry: the expression of 15 proteins(ubiquitin-conjugating enzyme E2 variant 1,taf6 RNA polymerase Ⅱ,nuclear autoantigen,Enoyl-Coenzyme A(Coa),nitrilase homolog 1,thioredoxin-related transmembrane protein 2,erbB3 The expression level of 1,unnamed protein product,fas(TNFRSF6)binding factor 1)was increased by EGF stimulation,and PKG Ⅱeffeciountly inhibitied the increases;the expression of 2 proteins(marvelD3 and annexin ⅡI)was decreased the by EGF treatment,and PKG Ⅱ efficiently reversed the inhibitory effect of EGF.To confirm the two-dimensional electrophoresis and mass spectrometry identified changes of protein expression,4 proteins(Prdx3、UBE2V1、FUBP1 andMarvelD3)of the above 17 proteins were seleted and their expressions were detected by Western blotting.The results showed that EGF treatment causedincrease of expression of Prdx3,UBE2V1 and FUBP1,and caused decrease of MarvelD3 expression in AGS and HGC-27 cells;PKG Ⅱ efficientlyinhibited the above changes of protein expressions caused by EGF treatment.The study also chosed MarvelD3 and applied RNA interference technologyto nvestigate its role in inhibiton of PKG Ⅱ on EGFR mediated activities.Thre results showed that PKG Ⅱ down-regulated EGF-induced expression of MarvelD3,significantly inhibited the enhancement of EMT and migration ability of EGF-stimulated tumor cells.For protein phosphorylation,we applied phosphorylation protein enrichment kit to isolate and enrich phosphorylated proteins from AGS cells.The phosphor-protien examples were subjected to two-dimensional electrophoresis and mass spectrometry and 5 phospho-proteins(Hirip3,HBO1,Basonuclin 2,LSFR1 and PEA15)were indentified.The phosphorylation level of three proteins(Hirip3,HBO1 and Basonuclin2)was increased by EGF treatment and PKG Ⅱefficiently inhibited the increase;the phosphorylation levelof 2 proteins(LSFR1 and PEA15)was decreased by EGF treatment and PKG Ⅱ efficiently reversed the inhibitory effect of EGF.To confirm the results two-dimensional electrophoresis and mass spectrometry,IP-Western blottingwas applied to detect the tyrosine phosphorylation of Hirip3 and HBO1 in gastric cancer cell lines AGS and HGC-27 treated with EGF and/or PKG Ⅱ.The results showed that EGF could significantly stimulate tyrosine phosphorylation of Hirip3 and HBO1,and PKG Ⅱcould suppress this change,confirming the inhibitory effect of PKG Ⅱ on EGF/EGFR induced protein phosphorylation.The study also chosed Hirip3 to explore the mechanism by which PKG Ⅱ inhibit EGF induced protein phosphorylation.Nuclus and cytoplasma separation method was appllied to dectet the localization of EGFR within the cells.The results showed that in AGS and HGC-27 cells,PKG Ⅱcould inhibit EGF-induced nuclear translocationof EGFR.Co-IP results showed the binding between Hirip3 and EGFR.These results suggested that EGFR bound directly to Hirip3 and phosphorylated it and PKG Ⅱ inhibited the process by blocked the nuclear translocation of EGFR.This study also primay explores the significance of PKG Ⅱ inhibition on EGF/EGFR induced protein phosphorylation: since HBO1 and Hirip3 were able to modulate histone modification(methylation,acetylation),we used Western blotting to detect the changes of histone expression levels in the downstream of these two proteins.The results showed thatn in AGS cells,PKG Ⅱcould significantly reduce the expression of H3135,H3K27me3 and H4K5 ac in the downstream of Hirip3 and HBO1 caused by EGF treatment.In HGC-27 cells,EGF treatment increased the expression of H3K56 ac and H4K5 ac,which were downstream proteins of Hirip3 and HBO1 respectively.At the same time,it was found that PKG Ⅱ significantly inhibitedEGF treatmentinduced expression of H3K4me3,a major transcriptional activator marker.In summary,the results of this study showed that PKG Ⅱinhibited the EGF treatment induced protein expressions,and also reversed the change of protein phosphorylation caused by EGF treatment.This confirmed the inhibition of PKG Ⅱ on EGFR activation mediated cellular bioactivities at protein expression and protein phosphorylation level,the last event of signalings,providing further evidence to indentify PKG Ⅱ as anti-cancer factor.