Smurf1 Enhances The Activity Of PDK1 By Neddylation

ObjectiveThe phosphoinositide 3-kinase(PI3K)/ protein kinase B(PKB/Akt)signaling pathway is essential in cellular process regulation.Akt is the key member in the regulation of this pathway.3-phosphoinositide-dependent protein kinase-1(PDK1)is upstream of Akt and could phosphorylate it at Thr308 to activate its kinase activation.However,the post-translation modifications of PDK1,other than phosphorylation,is rarely reported.Herein,We explored the mechanism of Neddylation modification of PDK1(3-phosphoinositide-dependent protein kinase 1)and its effect on Akt phosphorylation kinase activity.MethodsCod-IP(Co-IP)and Western blot(WB)were used to analyze the Nddylation mechanism of PDK1.The ubiquitination-like modification experiment screened Smurf1 to promote PDK1 Neddylation modification.Site-directed mutagenesis and co-immunoprecipitation and Western blotting were used to determine the interaction between PDK1 and Smurf1.The PDK1 / Akt signaling pathway can function through EGF or FBS stimulation.In normal MEF cells,epidermal growth factor stimulation induced phosphorylation of Akt was up-regulated at 5 min,for 1 h without significant decrease.Here,we selected Smurf1 knockout MEF cells and Smurf1 wild-type MEF cells,EGF stimulation observed changes between the two.Resultswe revealed PDK1 is a new substrate of NEDD8.We verified Smad ubiquitylation regulatory factor-1(Smurf1),a HECT-type E3,is the NEDD8-E3 of PDK1.Smurf1 interacts PDK1 with its WW domain and neddylate its kinase domain,this could increase the catalytic ability of PKD1.Knockdown of Smurf1 or blocking the neddylation pathway with MLN4924 would decrease the phosphorylation of Akt and desensitize cell to extracellular signaling.ConclusionsSmurf1 promotes PDK1 Neddylation modification and enhances its Akt phosphorylation activity.Our study provides a new understanding of PDK1 regulation and revealed a new crosstalk between PI3K/Akt pathway and neddylation pathway.