The Role Of G?i Inhibitory Subunits G?i1 And G?i3 In The Activation Of Akt-mTORC1 Pathway By Ciliary Nerve Growth Factor

Objective:1.To study the role of G protein inhibitory subunit G?i1 and G?i3 in activating Akt-mTORC1 pathway and increasing the phosphorylation level of Gab1,Erk and GSK-3? by ciliary nerve growth factor(CNTF).2.The role and mechanism of G?i1 and G?i3 in rotenone and 6-OHDA-induced cell injury.Methods:The wild type mouse embryonic fibroblasts(WT MEFs),G?i1 and G?i3 double knockout MEFs(DKO MEFs),single knockout G?i1,G?i3 or G?i2 MEFs(G?i1-KO MEFs,G?i3-KO MEFs,G?i3-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-3?(9S),GSK-3?,pmTOR,mTOR,pS6K(389T)and pS6(235/236S)were analyzed by western blotting.The G?i1 and G?i3 in MEFs were knocked down by shRNA.The expression of G?i1 and G?i3 were restored by reintroducing G?i1 and G?i3 gene into DKO-MEFs.Western blotting was used to detect Akt-mTORC1 pathway and the level of pErk1/2,pGab1,pGSK-3? to confirm the important role of G?i1 and G?i3 in CNTF activating Akt-mTORC1 pathway,Erk1/2,Gab1 and inhibiting GSK-3?.SK-N-SH was treated with rotenone and CNTF,and the cell viability was detected by MTT assay.The expression of G?i1 or G?i3 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 G?i1 or G?i3 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 G?i1 and G?i3 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-3?(9S),pmTOR,pS6K(389T),pS6(235/236S),and p Gab(627Y)induced by CNTF were decreased siganificantly in DKO MEFs.2.Lacking G?i1 or G?i3 genes inhibited the activation of Akt-mTORC1 signaling or the phosphorylation of Erk,Gab1 and GSK-3?induced by CNTF,and the effect of G?i3 protein more importing than G?i1,however,G?i2 protein was not necessary.3.The p Akt(473S),pAkt(308T),pErk1/2,pGSK-3?(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 G?i1 and G?i3 protein could reduce the damage of rotenone,and decrease the level of pP38,pJNK and pAMPK.Conclusion: G?i1,G?i3 and Gab1 play an important role in activation of Akt-mTORC1 pathway and phosphorylation of Erk and GSK-3?,Gab1 by CNTF.And Gab1 is located downstream of G?i protein and upstream of Akt signal protein.Lacking G?i1 and G?i3 protein could protect cells from rotenone and 6-OHDA.The probable mechanism is that the deletion of G?i1 and G?i3 attenuates the activation of P38,JNK and AMPK by rotenone and 6-OHDA.