Identificaiton Of The Ubiquitination Sites And Ubiquitin Ligase E3of C-Maf

Part1The lysine sites of c-Maf for ubiquitination[Objective]To identify the specific lysine residues responsible for c-maf ubiquitinaiton andstability.[Methods]1. The wild-type (WT)-c-Maf and a series of c-Maf mutants with lysine (K) arginine (R) were made by site-directed mutagenesis. These mutants included singlewild type lysine residue or single lysine mutant ones, as well as c-Maf all lysinemutations or multiple lysine mutations. The mutants were transfected into the HEK293Tcell line. Thirty-six hrs later, the cells were treated with MG132for4hrs before celllysates were extracted. The Western Blotting was used to measure the changes of thec-Maf protein.2. The c-Maf and the key mutants were transfected into the HEK293T cell line.Thirty-six hours later, the cells were treated with MG132for4hrs before cell lysateswere extracted. Proteins were then subject to immunoprecitation (IP) and WesternBlotting analysis.3. The c-Maf and the key mutated genes were constructed into the pcDNA-EGFPvector. These plasmids were then transfected into HEK293T cells. Twenty-four hourslater, cells were subject to confocal microscopy analysis to visualize their subcellularlocalization.4. The CCND2-Luc and WT-c-Maf or the mutants were cotransfected into theHEK293T cell line. Thirty-six hrs later, the Luciferase activity was detected. [Results]1. The results showed that a single lysine residue was not sufficient for c-Mafubiquitination, and a single lysine mutant was not able to prevent c-Maf ubiquitination.Further studies found that c-Maf ubiquitination was mediated by multiple lysineresidues, at least two lysine residues were required, such as K(85,350). However,K(85,350)R could not completely block c-Maf ubiquitination.2. The ubiquitination level of c-Maf modulated the cellular discribution. Confocalmicroscopy analysis indicated that wild type c-Maf protein was evenly expressed in thenuclei, but the lysine-free mutant c-Maf protein formed the aggregates and wasdistributed in both the nuclei and cytoplasmic compartments.3. Ubiquitination modulated the biological function of c-Maf. In the analysis of3mutants including K(85,350), K(85,350)R and M14by using WT as the control. TheWT, K(85,350), and K(85,350)R showed the similar effects in transactivating cyclin D2promoter, but the M14mutant showed a higher mudolating activity toward cyclin D2protmoter.[Conclusions]By a series of screening on the lysine residues in c-Maf protein, a single lysinemutant (K to R) was not sufficient for c-Maf ubiquitnation. Multiple lysine residueswere required to mediate c-Maf ubiquitination and turnover. Among these key resides,co-existence of K85and K850is important for c-Maf ubiquitination. The ubiquitinationlevel of c-Maf modulated the cellular discribution and its biological function.Part2c-Maf degradation via lysosomes[Objective]The M14with the lysine residue mutations (14K to14R) also can be degraded. Wewondered whether c-Maf stability was also mediated by other avenues, such as thelysosome pathway. Therefore, we examined wheather c-Maf can be degraded in thelysosmes by using the lysosome inhibitors.[Methods]1. The WT and M14c-Maf were transfected into the HEK293T cells. After48hrs, the cells were treated with the CHX for a certain perioid before being subject to lysisand applied for Western Blotting to measure the protein abundance of c-Maf.2. The WT and M14c-Maf were transfected into the HEK293T cells. After48hrs,the CHX alone or combination with the proteasome inhibitor (Bortezomib) and differentlysosome inhibitors (chloroquine, amino chloride) were added. And then, cells werecollected for protein extracts to measure c-Maf protein levels at0,0.5,1,2,4,6,8, and12hrs after CHX treatment.[Results]1. The WT c-Maf was degraded markedly in0-8hrs after treatment with CHX,while the M14was weakly degraded although M14was also degraded in8-12hrs aftertreatment with CHX, suggesting M14degradation was in a slower rate than its WTcounterpart.2. Both the proteasome inhibitors and lysosme inhibitors could slow down thedegradation of WT c-Maf. And the proteasome inhibitors in combination with lysosomeinhibitors were better than the single one. But only the lysosme inhibitors could slowdown the degradation of M14.[Conclusions]The c-Maf can be degraded via both the proteasome and lysosome pathways.Part3Identificaiton of the ubiquitin ligase E3of c-Maf[Objective]The above studies have clearly stated that c-Maf can be modified by multipleubiquitination, but its specific E3ligase was not identified yet. The prupose of thissection is aimed to identify this E3based on a luciferase screening and a co-IPcoupled-LC/MS/MS strategy.[Methods]1. Individual E3plasmids were transfected into NIH3T3cells which stably expressc-Maf and CCND2promoter-driven luciferase (CCND2-Luci) by lipofectamine2000.After48hrs, luciferase activity from each treatment was detected. 2. To further identify E3ligase for c-Maf, the possible E3s were cotransfected withc-Maf and ubiquitin into NIH3T3, followed by a measurement on c-Maf protein.3. LC/MS/MS analysis of proteins involved in c-Maf ubiquitination. The c-Mafand ubiquitin plasmids were cotransfected into HEK293T cells. Thirty-six hours later,cells were treated with MG132for4hrs. Then the proteins interacting with c-Maf werepulled down by anti-HA agarose beads. Finally, those proteins were identified byLC/MS/MS. The proteins related with the ubiquitination of c-Maf were obtained byDAVID clustering and KEGG pahway analysis.4. The HA-c-Maf, ubiquitin and Flag-E3were cotransfected into the HEK29T cells.After48hrs, proteins were co-IPed by anti-HA or anti-Flag agarose beads. Thewestern-blotting method was used to detect interacting protiens.5. The pEGFP-c-Maf and Flag-E3were co-transfected into HEK29T cells. Theimmunofluorescence was performed by anti-Flag antibody to localize the E3in the cells.The localization of c-Maf and E3were analysed by a laser confocal microscopy.[Results]1. By preliminary screening of the E3library,16possible E3s were narroweddown.2. cMul was singled out from the16possible candidates. It significantly promotedthe degradation of c-Maf.3. The results of LC/MS/MS showed that cMul interacted with c-Maf in HEK293Tcells and also found some other proteins related with ubiquitination of c-Maf.4. cMul was co-localized with c-Maf as observed by confocalfluorescentmicroscopy.[Conclusions]cMul, a probable E3of c-Maf, was identified via E3screening and LC/MS/MSstudy. cMul induces c-Maf degradation in a concentration-dependent manner. Physically,cMul is colocalized with c-Maf.ConclusionsIn the present study, we found c-Maf ubiquitination is mediated with multiple lysine residues, of which K85and K350were proved to play a key role in theubiquitination process of c-Maf protein. We also identified the E3ligase of c-Maf—cMul. These findings will help for in-depth understanding c-Maf biological activity andits role in the pathophysiology of multiple myeloma.