The Role And Mechanism Of PTEN-GSK3?-MOB1 Axis Regulating Neuronal Neurite Outgrowth

Objective: To investigate the role of PTEN-GSK3?-MOB1 axis in regulation of neurite outgrowth,and identify the phosphorylation site and ubiquitylation mediated by GSK3? at MOB1,which might provide the new strategy for efficient targeted drug screening and combined therapy after spinal cord injury(SCI).Methods:(1)Expression profile of PTEN and MOB1 protein during the development of mouse spinal and brain were determined by Western blot;(2)We used mouse neuroblastoma Neuro2 A and rat pheochromocytoma PC12 cell lines as in vitro research models for the study of the effect of PTEN/MOB1 on the neurite outgrowth by gene transfection,Real-time PCR,Western blot and immunofluorescence;(3)We explored the mechanism of PTEN regulation of the stability of MOB1 protein using Western blot;(4)We identified the phosphorylation sites and ubiquitylation mediated by GSK3? at MOB1 using point mutant techniques,Western blot and IP assays;(5)We studied the impact of gene silencing of MOB1 on neurite outgrowth and functional recovery after SCI in the PTEN inhibition background.Results:(1)During the development of mouse spinal cord and brain,PTEN was negatively correlated with the expression pattern of MOB1 protein.In addition,PTEN and MOB1 expression patterns were negatively correlated in muridae cells;(2)PTEN knockdown promoted the neurite outgrowth and increased the level of MOB1 protein in PC12 and Neuro2 A cells,and the level of MOB1 m RNA did not change.Three specific pharmacologic inhibitors of PTEN-PI3 K downstream pathways could reversed the effect of PTEN knockdown on the protein level of MOB1;(3)Overexpression of MOB1 A and MOB1 B significantly promoted neurite outgrowth in PC12 cells;(4)We treated PC12 cells with a GSK3? inhibitor,Li Cl,along with a protein synthesis inhibitor,CHX and found that Li Cl stabilized MOB1 protein in the presence of CHX.PTEN knockdown significantly increased the half-life of MOB1.In addition,MOB1 expression was considerably stable in MEF GSK3?-/-cells with CHX;(5)IP results showed that MOB1 could bind to GSK3? in NIH3T3 cells,and exogenous MOB1 A and MOB1 B could also bind to GSK3?.Meanwhile,the results showed that MOB1 was degraded via the ubiquitin-proteasome system(UPS),and this effect would be regulated by GSK3?;(6)Analysis of the MOB1 coding sequence identified three motifs that resemble the GSK3? target sequence: T76,T130 and S146.We then generated T76 A,T130A,and S146 A mutants and found that the S146 A mutant had a notably longer half-life than wild-type(WT),while T76 A and T130 A mutants were not significantly changed.Consistent with its longer half-life,the S146 A mutant had the lowest ubiquitination level among the MOB1 mutants and WT;(7)In contusion model of T9 spinal impact injury in mice,we injected lentivirus sh RNA vectors that targeted PTEN and MOB1 into the injured spinal cord of mice and waited 6 weeks.The results showed that PTEN knockdown promoted neurite outgrowth and functional recovery in spinal cord contusion mice by immunofluorescence staining,Footprint analysis and BMS Score.However,the above-mentioned phenomenon would be reversed by MOB1 knockdown.Conclusions:(1)PTEN was negatively correlated with the expression of MOB1 protein during the development of moues CNS;(2)MOB1 promoted neurite outgrowth in PC12 cells;(3)PTEN regulated MOB1 protein stability by activation of the GSK3? which bound and phosphorylated MOB1 at Ser146 site and then degraded ubiquitylated MOB1 via the UPS;(4)Lentiviral-mediated silencing of PTEN promoted neurite outgrowth and functional recovery after SCI and this effect was reversed by down-regulation of MOB1.