Proteasome inhibition augments conjugate formation between JunB and FAT10

To date, ubiquitin and FAT10 are the only ULMs that have been described to tag proteins for the degradation through the 26S proteasome (Hershko, 1983; Hipp et al., 2005).

Like ubiquitin, FAT10 bears a free di-glycine motif at the C-terminus, which mediates the conjugation to target proteins. To elucidate, if the JunB-FAT10 conjugate is degraded via the proteasomal pathway, a MYC-FLAG- tagged JunB construct was expressed together with HA-FAT10 or together with HA-FAT10 and HIS-TRIM11 in HEK293 cells. Transfected cells were pre-treated with the proteasome inhibitor MG132 (10 µM), 6 h before cell lysis, or left untreated. Moreover, to ensure that conjugate formation of JunB and FAT10 consists of an isopeptide-linkage, we co-expressed JunB-MYC-FLAG, HIS-TRIM11 together with HA-FAT10ΔGG, which served as a negative control, since HA-tagged-FAT10ΔGG is not capable to form an isopeptide linkage to lysine residues on specific substrates.

Results

103 (c)

(a)

(b)

104

Figure 22: Conjugate formation of FAT10 and JunB in vivo.

Co-immunoprecipitation of JunB-MYC-FLAG, HA-FAT10, HA-FAT10ΔGG and HIS-TRIM11 as indicated. HEK293 cells were transiently transfected with pCMV6-JunB-MYC-FLAG, pcDNA3.1-HA-FAT10, pCDNA-3.1-HA-FAT10ΔGG and pcDNA3-HIS/-A-TRIM11. Cells were treated either with/without the proteasome inhibitor MG132 (10 µM) for 6 hours. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). After immunoprecipitation against the HA-Tag of HA-FAT10 and HA-FAT10ΔGG with anti-HA-agarose, samples were separated on 4-12% Bis-Tris SDS gels and subjected to Western blot analysis with a horseradish peroxidase (HRP)-conjugated HA-reactive antibody (a). The expression of HIS-TRIM11 was detected with a peroxidase (POX) conjugated polyhistidine-reactive antibody (b). The expression of JunB-MYC-FLAG was detected with a FLAG-reactive antibody conjugated to horseradish peroxidase (HRP). Conjugate formation of HA-FAT10 and JunB-MYC-FLAG could be detected after immunoprecipitation against the HA-Tag of FAT10 and Western blot analysis against the FLAG-tag of JunB (c). The plasmid content in all transfections was balanced with empty expression vector pcDNA3. β-actin served as a loading control.

24 h after transfection, immunoprecipitation with anti-HA agarose was performed to accumulate FAT10-conjugated proteins. Analysis by Western blotting revealed, that at the height of ~66 kDa a double band appears the size of which equates to a monoFAT10ylated form of JunB (as shown before in Figure 21), detected through a peroxidase-conjugated FLAG-reactive antibody against the FLAG-Tag of JunB (Figure 22 (c), lanes 4-7). The appearing double band led to the assumption, that different forms of JunB are modified with FAT10. Whether this is a phosphorylated or other modified form of JunB remains hitherto unclear. The amount of JunB-FAT10 conjugates could be augmented significantly, when cells were pre-treated with MG132 for 6 hours before cell lysis, suggesting an involvement of the proteasome for conjugate degradation. No conjugation formation was detectable when JunB was co-transfected together with FAT10ΔGG (Figure 22 (c) lane 8-11).

This result strengthens the presumption, that FAT10 becomes isopeptide linked to an ε-lysine residue of JunB, since FAT10ΔGG, lacking the di-glycine motif is not capable to form this linkage. Co-transfection of JunB together with either FAT10 or FAT10∆GG decreased the amount of FAT10 and FAT10∆GG, while treatment with MG132 recovered the effect (Figure 22 (a) lane 3, 4 and lane 8, 9). Moreover, co-transfection of TRIM11 highly decreased the amount of JunB, FAT10 and FAT10∆GG (Figure 22 a), b) and c) lane 6-7).

This effect could in turn partially be rescued with MG132 treatment (Figure 22 a-c). Further, conjugate formation of JunB and FAT10 was almost completely abolished, when TRIM11 was co-expressed, which argue in the first instance against the hypothesis, that TRIM11 is a FAT10 specific E3 ligase with JunB as substrate. It rather let suppose a role for TRIM11 in controlling JunB and JunB conjugate stability. There is a strong hint that FAT10 modification serves as a degradation signal, because the conjugate was accumulating after proteasome inhibition.

Results

105 6.2.5 JunB has no influence on the degradation rate of FAT10

Attachment of FAT10 causes the rapid degradation of long-lived proteins, which is dependent on the 26S proteasome (Hipp et al., 2005). In contrast to ubiquitin, FAT10 has a relatively short half-life because it is also subject to proteasomal degradation in its monomeric form and, additionally, it is probably not recycled but instead degraded along with its substrates (Hanna and Finley, 2007). The turnover of FAT10 was described to decrease rapidly after addition of cycloheximide (Raasi et al., 2001) showing that the half-life of FAT10 is ~1 hour. To show as a control that JunB expression does not alter FAT10 degradation rate, we next investigated in a cycloheximide chase if JunB interaction with FAT10 would have functional relevance for the degradation of FAT10 in vivo. The compound cycloheximide inhibits protein synthesis by blocking translation elongation and therefore can be used in time-course experiment followed by Western blotting of the cell lysates for the protein of interest to determine the half-life of proteins. HEK293 cells were transfected with pCMV6-JunB-MYC-FLAG and pcDNA3.1-HA-FAT10 constructs and cells were treated with cycloheximide (50 µg ml^-1) for the indicated time points. Six hours before harvesting MG132 (10 μM) was added, as indicated. Whole-cell lysates were either directly immunoblotted 24 h post-transfection with an anti-HA reactive antibody or subjected to co-immunoprecipitation assays, using anti-HA agarose followed by SDS-PAGE and Western blotting with a anti-HA antibody (Figure 23).

Figure 23: Co-expression of JunB has no influence on the degradation rate of FAT10.

HEK293 cells were transiently co-transfected with pCMV6-JunB-MYC-FLAG and pcDNA3.1-HA-FAT10 plasmid.

Before cell lysis, cells were treated with the proteasome inhibitor MG132 (10 µM) for 6 hours and with cycloheximide (50 µg ml^-1) at different time points, as indicated. After immunoprecipitation against the HA-Tag of FAT10 with anti-HA agarose, samples were separated on 4-12% Bis-Tris SDS gels and subjected to Western blot analysis using a directly peroxidase-linked anti-HA mAb to evaluate FAT10 expression. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). β-actin served as a loading control. Graphs show the quantification of HA-FAT10 in the cells. HA-FAT10 ECL signals of all experiments were quantified with Quantity One Software (BioRad) and mean values ± SEM of five independent experiments are depicted as relative expression to the ECL signal of HA-FAT10 transfected cells without cycloheximide treatment, which was set to unity.

106 The quantitative analysis revealed a half-life for FAT10 of approximately 1 h (Figure 23) when co-expressed together with JunB. In accordance with a previous report (Raasi et al., 2001) the half life of FAT10 is 1 h. After 2.5 h the amount of FAT10 decreased to 25 % and 5 h after CHX treatment only ~10 % FAT10 remained left. Treatment with MG132 led to an accumulation of monomeric FAT10 (~150 %) by preventing degradation via the proteasome.

Taken together, these results indicate that co-expression of JunB did not change the degradation rate of FAT10.

6.2.6 Co-expression of FAT10 hardly affects the degradation of unconjugated