The Role Of Gαi Inhibitory Subunits Gαi1 And Gαi3 In The Activation Of Akt-mTORC1 Pathway By Ciliary Nerve Growth Factor

Objective:1.To study the role of G protein inhibitory subunit Gαi1 and Gαi3 in activating Akt-mTORC1 pathway and increasing the phosphorylation level of Gab1,Erk and GSK-3β by ciliary nerve growth factor(CNTF).2.The role and mechanism of Gαi1 and Gαi3 in rotenone and 6-OHDA-induced cell injury.Methods:The wild type mouse embryonic fibroblasts(WT MEFs),Gαi1 and Gαi3 double knockout MEFs(DKO MEFs),single knockout Gαi1,Gαi3 or Gαi2 MEFs(Gαi1-KO MEFs,Gαi3-KO MEFs,Gαi3-KO MEFs).The MEFs were treated with cortical nerve growth factor.The pGab1(627Y),Gab1,Akt1/2/3,pAkt(473S),pAkt(308T),pErk1/2,Erk1/2,pGSK-3β(9S),GSK-3β,pmTOR,mTOR,pS6K(389T)and pS6(235/236S)were analyzed by western blotting.The Gαi1 and Gαi3 in MEFs were knocked down by shRNA.The expression of Gαi1 and Gαi3 were restored by reintroducing Gαi1 and Gαi3 gene into DKO-MEFs.Western blotting was used to detect Akt-mTORC1 pathway and the level of pErk1/2,pGab1,pGSK-3β to confirm the important role of Gαi1 and Gαi3 in CNTF activating Akt-mTORC1 pathway,Erk1/2,Gab1 and inhibiting GSK-3β.SK-N-SH was treated with rotenone and CNTF,and the cell viability was detected by MTT assay.The expression of Gαi1 or Gαi3 in SK-N-SH cells was knocked down by lentivirus with shRNA.MTT assay was used to detect the activity of SK-N-SH with knockdowned Gαi1 or Gαi3 after treating with CNTF and rotenone.Western blotting was used to analysis the changes of related signal proteins to further study the protective mechanism of Gαi1 and Gαi3 in CNTF proctectting SK-N-SH treated with rotenone.Rotenone and 6-OHDA were used to treat WT MEFs and DKO MEFs Respectively,the cell viability was analysis by MTT assay,and the p P38,pJNK and pAMPK were detected by Western Bliotting.Results:1.The pAkt(473S),pAkt(308T),pErk1/2,pGSK-3β(9S),pmTOR,pS6K(389T),pS6(235/236S),and p Gab(627Y)induced by CNTF were decreased siganificantly in DKO MEFs.2.Lacking Gαi1 or Gαi3 genes inhibited the activation of Akt-mTORC1 signaling or the phosphorylation of Erk,Gab1 and GSK-3βinduced by CNTF,and the effect of Gαi3 protein more importing than Gαi1,however,Gαi2 protein was not necessary.3.The p Akt(473S),pAkt(308T),pErk1/2,pGSK-3β(9S),pS6K(389T)and pS6(235/236S)induced by CNTF were damaged in Gab1-ko MEFs.4.CNTF protected SK-N-SH from rotenone.Lacking Gαi1 and Gαi3 protein could reduce the damage of rotenone,and decrease the level of pP38,pJNK and pAMPK.Conclusion: Gαi1,Gαi3 and Gab1 play an important role in activation of Akt-mTORC1 pathway and phosphorylation of Erk and GSK-3β,Gab1 by CNTF.And Gab1 is located downstream of Gαi protein and upstream of Akt signal protein.Lacking Gαi1 and Gαi3 protein could protect cells from rotenone and 6-OHDA.The probable mechanism is that the deletion of Gαi1 and Gαi3 attenuates the activation of P38,JNK and AMPK by rotenone and 6-OHDA.