Function Of P38?/? In The Formation Of Ployubiquitinated Protein Aggresome

Objective:To study the function of p38?/? in the formation of cellular polyubiquitinated protein aggresome during the proteasome inhibition.Methods:?To test whether the p38?/? were activated during the cellular proteasome inhibition,western blotting using the antibody which specially recognizes the p38 proteins with dual phosphorylation at residues Thr180/Tyr182 was applied to detect the phosphorylation of p38?/? in the wild type AD293 treated by proteasome inhibitor MG132.?CRISPR/Cas9 was applied to generate the AD293 p38?/? knock-out cell lines,and western blotting using the antibodies which specially recognize p38?/? respectively were applied to determine whether the KO cell lines were generated successfully.The cDNAs of p38?/? were tagged with Flag tag respectively and then cloned into lentivial vectors,and using the lentiviral expression system we generated rescue cell lines which stably express Flag-p38?/? based on the KO cell lines.?The p38?/? KO cell lines and Flag-p38?/? rescue cell lines were treated with MG 132.The cellular polyubiquitinated proteins were labeled by UbiK48 antibody,and using the immunofluorescence technique with confocal microscopy the formation of aggresome in different cell lines were examined.?Using lentiviral expression system we generated the AD293 cell lines which stably express p38y(K56R)-Flag or p38?(K54R)-Flag,p38?/? and their interacted proteins were enriched by immunoprecipitation,separated by SDS-PAGE,and then identified by mass spectrum.?The vector used to express TPM1(pcDNA3.1(+)-TPM1-c3myc)was generated.The vector was transiently transfected into the AD293 p38?/? KO cell lines,using the immunofluorescence technique with confocal microscopy the formation of aggresome in the cells were examined.?Statistical analysis:t-test was applied to analyze the significance level.Results:?Compared with the non-treated cell,the phosphorylation level of p38?/? in the cell treated by MG 132(2uM,16hr)were significantly enhanced,it is suggested that the p38?/? were activated during the proteasome inhibiton.?The endogenous p38?/? were detected by antibodies specially recognize p38?/? in the wild type AD293,p38?/? KO cell lines respectively,the western blotting showed that the p38?/? KO cell lines were generated successfully by the CRISPR/Cas9 technique.?The p38?/? KO cell lines were treated with MG132(2uM,16hr),and then immunostained by UbiK48 antibody,the formation of aggresome was observed by immunofluorescence with confocal microscopy.The results showed that compared with wild type AD293,the formation of aggresome in the KO cell lines were significantly reduced,the number of aggresome in p38? KO cell line is much less than that in p38? KO cell lines.Statistic datas showed that compared with the AD293,the numbers of aggresome were no less than 10%and 5%in p38?/? KO cell lines respectively,in the Flag-p38?/? rescue cell lines,the numbers of aggresome were reverted to 75%and 88%respectively.These results suggested that p38?/? indeed function in the formation of aggresome.?Using the AD293 cell lines which stably expressing p38?(K56R)-Flag or p38?(K54R)-Flag,the P38?/?-Flag and their interacted proteins were enriched by immunoprecipitation,separated by SDS-PAGE and identified by mass spectrum,One of the interacted proteins-TPM1 was identified as the potential substrate of p3 8?.?Overexpressing TPM 1 in p3 8? KO cell line,and then treated with MG132,the scattered polyubiquitinated protein aggregates but not aggresome can be observed in the cytosol,it seems that overexpressing TPM1 in p38y KO cell line cannot rescue the deficiency of aggresome formation.Conclusion:?In the condition of proteasome inhibiton,the cellular p38?/? were activated.?The deletion of p38?/?leads to the deficiency of aggresome formation,but overexpressing exogenous p38?/? can rescue the deficiency.?One potential substrate of p38?-TPM1 was obtained and identified by immunoprecipitation and mass spectrum.?Transiently overexpressing TPM1 can partially rescue the deficiency of aggresome formation in p38? KO cell line but not in p38y KO cell line.