Neddylation Inhibitor MLN4924 Inhibits Assembly Of Primary Cilia By Activating AKT1

Primary cilia act as a cellular receptor that protrudes from the surface of the cell membrane and is present in almost all types of cell surfaces in the body.There are a lot of proteins which are receptors of important signaling pathway located at the ciliary membrane,thus primary cilia can recognize extracellular signals and transmit them into the cell to regulate several pivotal physiological processes.The primail cilia play an indispensable role in the regulation of the developmental process of human body.AKT,also known as PKB or Rac,is a proto-oncogene that is an indispensable key survival protein in cells.AKT is a component of the PI3K-AKT-mTOR axis(phosphatidylinositol 3-kinase PI3Ks signaling pathway)and is activated by PI3Ks.It plays an indispensable role in many important physiological processes of cells.However,the role of PI3KjAKT in primary cilia has not been studied in depth.A few studies have shown that AKT 1/2 knockdown in maumalian cells can induce cilia assembly and inhibit cilia disassembly,whereas other studies showed that activation of the PI3K signal inhibits the formation of cilia.Therefore,the role of AKT1 in cilia remains elusive.To deternine whether MLN4924,a small molecular inhibitor of NEDD8 activating enzyme regUlates primary ciliogenesis,we established two cell culture models for cilia assembly and disassembly respectively.Using these two models,we found that compared with the control group,MLN4924 treatnent significantly reduced the number of ciliated cells.Because neddylation inhibition would inactive Cullin-RING ligases(CRLs)to cause accumulation of their substrates,we used Western blotting to measure the levels of few CRL substrates known to regulate eiliogesis.Unexpectedly,we found that MLN4924 treatment did not cause significant changes in the protein levels of the assembly key protein Cep97 or CP110.To further confirm their involvement,we used siRNA approach to knockdown Cep97 and CP 110 in Beas2B cells,but failed to rescue MLN4924-induced reduction of ciliated cell population.Previously,we have found that MLN4924 activates EGFR and its downstream AKT1 signal.We,therefore,pursued the involvement of AKT1 and found that indeed,MLN4924 inhibits cilia formation by inhibiting assembly and promoting disassembly via activating AKT1.In summary,we report here that in few cell models,MLN4924 inhibits ciliogenesis by a)inhibiting cilia assembly and b)promoting disassembly.Mechanistically,MLN4924 effect is mediated by activation of EGFR and AKT1 signals.Our study reveals how this neddylation inhibitor blocks ciliogenesis,and provides a foundation for future clinical trial of MLN4924 in the treatment of human diseases associated with overgrown cilia.