Interaction Study Between Virus Infection And SUMOylation Of PDK1

The 3-phosphoinositide-dependent protein kinase-1(PDK1)belongs to subfamily of the protein kinase A,G.PDK1 could phosphorylate lots of proteins,such as AKT,p90RSK,p70S6K,SGK and PKC.The activity of PDK1 plays viral role in a lot of cell function.However,the knowledge on the environmental cues affect PDK1 is still limit.In order to know the effection of virus infection on the activity of PDK1,we detect the affection of infection from influenza virus,IBDV and PCV2 on the PDK1.We found that virus infection could induce the modification of PDK1(the molecular mass is about 210KD)at 1,3,6,12hpi.Further study suggesting that IBDV infection modified PDK1 through VP3 and the modification of PDK1 resulted from SUMOylation.SUMOylation sites delete mutation results in the lack of modification bands of PDK1 confirmed the SUMOylation of PDK1.We also found that SUMOylation sites delete mutation lost the function on phosphrylating AKT and S6K.Since SUMOylation of PDK1 plays such viral role in life course,the molecular mechanisms involved in SUMOylation of PDK1 is very important.We useed PDK1 as a bait to fish the interaction protein with it.And we got a bands fished by PDK1(molecular mass=110 KD).The Mass Spectrometry analysis of this band indicated the VPS34.We confirm the direct interaction between VPS34 and PDK1 by CO-IP and pull down technology.Further study suggested that VPS34 negatively regulates the SUMOylation of PDK1.We found that VPS34 could destroy the interaction between PDK1 and UBC9 and subsequently inhibits the SUMOylation of PDK1.SUMOylation plays an important role in protein function.However,knowledge on regulating AKT by PDK1 SUMOylation is still unknown.Confocal analysis suggested that SUMOylation of PDK1 could promote the colocalization of PDK1 with AKT to membrane.CO-IP analysis suggested that SUMOylation of PDK1 promoted the interaction between PDK1 and AKT,and phosphorylating both of them.IBDV could destroy the interaction between VPS34 and PDK1 by binding of VP3 to VPS34 and subsequently promote the SUMOylation of PDK1.To map the key domain of VP3 in interaction between VP3 and VPS34,we found that CC3 domain of VP3 plays a key role in modifying PDK1 and phosophorylation of AKT.While the CC1 domain of VP3 could interact with Beclinl,the CC3 domain interact with VPS34.Thus,VP3 could activate AKT because of the interaction with VPS34,but not Beclinl.Therefore,VP3 induces autophagy is mainly through AKT pathway,but not Beclinl.We conclude here that virus infection regulates the activation of AKT via mediation of SUMOylation of PDK1 through the dynamic interaction between VPS34 and PDK1,which controls the infection progress of virus infection.