GCN2 Sense Na~+,K~+-ATPase Impairment And Couple Cells Survival And Death

Na+,K+-ATPase is an important ion transporter ubiquitously distributed in mammalian cell membrane.The function of which is to transport Na+ and K+ against a concentration gradient across the membrane,and maintain the Na+,K+ concentration gradient,membrane resting potential,osmotic balance,nutrient uptake,and other functions.Na+,K+-ATPase can also mediate signal transduction complex and affect the process of cell proliferation,differentiation and apoptosis.Recently,Na+,K+-ATPase is found to highly expressed in tumor cells,which raises the possibility of targeting Na+,K+-ATPase for cancer therapy.Na+,K+-ATPase inhibitors are naturally chemicals that can specifically bind Na+,K+-ATPase and suppress the activity.These inhibitors have been clinically used in treating heart failure or atrial arrhythmia,therefore,also known as cardiac glycosides.Interestingly,endogenous cardiac glycosides substances were also isolated and identified in human serum in recent studies,however,the concrete role of these endogenous substances remains largely unknown.Na+,K+-ATPase inhibitors can inhibit tumor growth at low concentrations,induce tumor cells apoptosis,enhance the sensitivity of tumor cells to chemotherapy or radiation therapy,which opens a new avenue for cardiac glycoside in cancer treatment.It is nothworthly to mention that in some normal or tumor cells,Na+,K+-ATPase inhibitors can stimulate cells growth,which limits the application of these chemicals as potential anti-tumor drugs.Therefore,delination of the regulatory mechanisms of cardiac glycosides on cell death and growth will be beneficial for development of more effective drugs.Tumor microenvironment plays important role for tumor growth.Under the condition of hypoxia and nutrient deprivation,tumor cells needs to rapidly adapt to these environment and gain growth advantage by triggering stress response.For example,tumor cells can utilize ER stress response and survive the disvantage via ATF4-mediated upregulation of many pro-survival moledules.Nothworthly,stress response signaling in tumor also offer many targets for cancer therapy.Although cardiac glycosides were found to be effective against many tumors,whether these small molecules take advantage of cellular response to exert their anti-tumor functions remain totally unknown.Our previous result showed that Na+,K+-ATPase inhibitors can trigger the translocation of nuclear stress protein HuR from nucleus to cytoplasm.HuR silencing,however,was able to increase tumor cells sensitivity towards Na+,K+-ATPase inhibitors,suggesting HuR exerts protective effect on Na+,K+-ATPase-induced cells apoptosis.In this study,Na+,K+-ATPase inhibitors tiggered HuR nuclear export and formed stress granules(SGs)in cells,which specifically recruited some pro-apoptotic genes,such as p21 mRNA into cells to protect cells from apoptosis.The occurrence of SG by Na+,K+-ATPase inhibitors were found to be dependent on GCN2-eIF2a phosphorylation signaling pathway by enhancing GCN2 and eIF2a interaction.GCN2(General control nonrepressed 2)is a widely distributed eIF2 alpha kinase that can be activated by uncharged tRNAs when amino acid is deficient.Owing to nutrient deprivation in tumor microenvironment in vivo,GCN2-eIF2a-ATF4 pathway is crucial to maintain metabolic balance in tumor cells.Theroretically,GCN2 is supposed to have similar protective effect as that of HuR,but the results obtained in this study is totally opposing,because tumor cells apoptosis induced by Na+,K+-ATPase inhibition is well-correlated with the expression level of GCN2 in tumor cells.Further,Na+,K+-ATPase inhibitors-induced cells apoptosis was abrogated in cells when GCN2 expression was silenced.Therefore,we presume that GCN2 has dual functions in regulating tumor cells survival and death.To understand the mechanism of GCN2 in regulating cells destiny,we found GCN2 degradation is mediated by ubiquitin.Interestingly,β-arrestinl/2,as a scaffold protein,can recruit E3 ligase RSP5 into GCN2 and form a ternary complex to control the basal level of GCN2 at a relatively low expression,but in the presence of ouabain treatment,this ternary complex was disrupted leading to elevated level of GCN2,which facilitate cells apoptosis.Finally,we identified that ouabain was able to trigger GCN2 phosphoylation at T899,by which the interaction between b-arrestins and GCN2 was impaired.Since GCN2 phosphorylation at T899 has both effects of SG formation and apoptotis-enhancing effects,we examined how these two functions of GCN2 switch in ouabain-induced apoptosis.As a result,we found GCN2 phosphorylation-mediated SG formation is mainly responsible for preventing cells from mild or transient stress of Na+,K+-ATPase impairment,however,as the intensity or duration of Na+,K+-ATPase impairment increased,GCN2 phosphorylation switched its pro-survival function to apoptosis enhancement by increasing GCN2 protein level.Finally,we also found that when the apoptotic program was initiated,dephosphorylation of GCN2 occurred to reduce its pro-survival effect,and meanwhile,recruited more dephosphorylated GCN2 into proteosome for degradation via forming the ternary complex.In sum,we described for the first time GCN2 phosphorylation was able to couple cells survival and death via SGs formation and ubiquitin-mediated degradation under Na~+,K~+-ATPase impairment.Furthermore,by using Na~+,K~+-ATPase inhibitors,we identified the novel regulatory mechanisms of stress response in tumor cells,which offers new targets for cancer therapy.