CHX data reveal a role for proteasome dependent degradation of the conjugate between JunB and FAT10

The in vivo data provided evidence that FAT10 becomes stably attached to JunB (see 6.2.3) and proteasome inhibition led to an accumulation of the conjugate (see 6.2.4). We could also show, that neither FAT10 (see 6.2.5) nor JunB turnover (6.2.6) is noticeably altered in its monomeric form, when not conjugated to each other. Since FAT10 is degraded by the 26S proteasome and targets FAT10-linked proteins for proteasomal degradation (Hipp et al., 2004; Schmidtke et al., 2009), we investigated in cycloheximide-chase experiments, if the turnover rate of the conjugate between JunB and FAT10 changes over time and is caused through proteasomal degradation. Moreover, the in vivo data already indicated that FAT10 becomes also non-covalently linked to JunB (see chapter 6.2.3 and 6.2.4.).

Hence, we next aimed to determine the turnover rate of covalently and non-covalently linked JunB and FAT10.

For this purpose, HEK293 cells were transiently transfected with MYC-FLAG-tagged JunB and HA-tagged FAT10 constructs and treated with cycloheximide (50 µg ml^-1) for 2.5 and 5 h, or left untreated. Six hours before cells were harvested, 10 μM MG132 was added to inhibit the proteasome. 24 h after transfection, whole-cell lysates were subjected to co-immunoprecipitation assays using anti-HA agarose, followed by SDS-PAGE and Western blotting with an anti-FLAG reactive antibody.

Results

109

Figure 25: CHX-chase: Determination of JunB-FAT10 conjugate turnover rate

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 h and with cycloheximide (50 µg ml^-1) at different time points, as indicated. Whole cell lysates were subjected to Western blot analysis with a FLAG-reactive antibody coupled to HRP. After immunoprecipitation against the HA-Tag of FAT10 with anti-HA agarose, samples were subjected to Western analysis using a directly horseradish peroxidase (HRP) -linked anti-FLAG mAb to evaluate conjugate formation. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). β-actin served as a loading control. ECL signals of all experiments were quantified with Quantity One Software (BioRad). A representative blot out of five independent experiments is shown. Mean values ± SEM of five independent experiments are depicted as relative expression to the ECL signal of HA-FAT10 JunB-MYC-FLAG transfected cells without cycloheximide treatment, which was set to unity.

Conjugate formation between FAT10 and JunB could be observed after immunoprecipitation against the HA-tag of FAT10 and immunoblotting against the FLAG-tag of JunB, apparent in the higher molecular weight double band at around 66 kDa (Figure 25). Regarding the conjugate stability of covalently linked FAT10 and JunB, a continuous decline during the indicated timepoints is detectable, which reveals a half-life of approximately 2.5-3 h compared to the much shorter half life of monomeric FAT10 (1 h) (Raasi et al., 2001).

The FAT10-JunB complex degradation could be rescued, when cells were pre-treated with the proteasomal inhibitor MG132 which led to a considerable accumulation of the conjugate (~180 %), suggesting a continuous degradation via the proteasome. Interestingly, non-covalently bound JunB was continuously degraded and revealed a similar rate of approximately 2.5 h. Combined with previous findings, these results suggest that the role of FAT10 is the rapid destruction of its target proteins via conjugation, either covalently or surprisingly also non-covalently, and subsequent degradation.

110 6.2.8 TRIM11 becomes degraded via the proteasome

The previous results did not provide clear evidence that TRIM11 acts either as a FAT10 or ubiquitin specific E3 ligase with JunB as substrate. We would expect, if TRIM11 is a special ubiquitin or FAT10 E3 ligase with JunB as substrate, conjugate formation to JunB would be enhanced, when the proteasome is inhibited at the same time. However, we could observe the opposite effect; overexpression of TRIM11 led to severe downregulation of JunB-Ub and JunB-FAT10 conjugates. There exists the possibility that TRIM11 is not an E3 ligase but rather a substrate for ubiquitin or FAT10 and attachment leads to to its proteasomal degradation. In this case a further E3 ligase could be involved to assign TRIM11 for proteasomal degradation.

So far, no protein half-life could be determined for endogenous TRIM11, because specific antibodies were missing. To characterize TRIM11 we investigated the turnover rate of TRIM11 in a cycloheximide chase with a new polyclonal TRIM11 antibody (Sigma), which reliably recognized endogenous and overexpressed TRIM11. To investigate, if TRIM11 is assigned to proteasomal degradation, cells were treated with or without MG132 for 6 h.

HEK293 cells were treated with cycloheximide (50 µg ml^-1) for 0, 2.5, 5 and 7.5 h, or left untreated. Six hours before cells were harvested, 10 μM MG132 was added.

Figure 26: Determination of endogenous TRIM11 turnover rate

HEK293 cells were treated before cell lysis with the proteasome inhibitor MG132 (10 µM) or left untreated for 6 hours and with cycloheximide (50 µg ml^-1) at different time points, as indicated. Samples were subjected to Western blot analysis using a polyclonal TRIM11 antibody (Sigma) to evaluate TRIM11 expression. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). β-actin served as a loading control. ECL signals of all experiments were quantified with Quantity One Software (BioRad). A representative blot out of three independent experiments is shown. Mean values ± SEM of three independent experiments are depicted as relative expression to the ECL signal of endogenous TRIM11 without cycloheximide treatment, which was set to unity.

Results

111 In HEK293 cells, a continuous decrease of endogenous TRIM11 for the indicated timepoints after cycloheximide addition could be observed and a protein half-life of approximately 7-8 hours was calculated (Figure 26). Only a slight rescue of TRIM11 could be measured when MG132 was added, which would argue against proteasomal degradation. However, cycloheximide treatment for more than 5 h led to increased cell death and additional apposition of MG132 enhanced the effect.

To compare the protein half-lifes of endogenous and overexpressed TRIM11 a second cycloheximide chase was performed.

HEK293 cells were transiently transfected with a TRIM11-FLAG construct and cycloheximide (50 µg ml^-1) was added for 2.5, 5, 7.5 or 10 h, or left untreated, before cell lysis. Moreover, cells were treated with or without MG132 for 6 h. After cell lysis, samples were boiled in Laemmli-Buffer containing 10% ß-mercaptoethanol followed by SDS-PAGE and Western blotting with a FLAG-reactive antibody.

Figure 27: Determination of ectopic expressed TRIM11 turnover rate

HEK293 cells were transiently transfected with pcDNA3-TRIM11-FLAG plasmid. Before cell lysis, cells were treated with the proteasome inhibitor MG132 (10 µM) for 6 hours and with cycloheximide (50 µg ml^-1) for different time periods, as indicated. After immunoprecipitation against the FLAG-Tag of TRIM11 with anti-FLAG agarose, samples were subjected to Western analysis using a directly coupled horseradish peroxidase (HRP) -linked anti-FLAG mAb to evaluate TRIM11 expression. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). β-actin served as a loading control. ECL signals of all experiments were quantified with Quantity One Software (BioRad). A representative blot out of three independent experiments is shown. Mean values ± SEM of three independent experiments are depicted as relative expression to the ECL signal of TRIM11-FLAG transfected cells without cycloheximide treatment, which was set to unity.

FLAG-tagged TRIM11 migrates as a double band with a size of 48 and 55 kDa. A continuous decline of overexpressed TRIM11-FLAG protein level is observable for the indicated timepoints and quantitative analysis revealed a half-life of approximately 5 h (Figure 27), which is much faster than for endogenous TRIM11.

112 Besides, treatment with MG132 completely blocked TRIM11-FLAG degradation and increased accumulation, which clearly substantiate an involvement of the proteasome for TRIM11-FLAG turnover.

These results clearly show that ectopically expressed TRIM11 is faster degraded than endogenous TRIM11, maybe due to excess of protein. Moreover, MG132 treatment leads to accumulation of TRIM11, which demonstrate an involvement of the proteasome in TRIM11 turnover.