Ubiquitin and FAT10 become isopeptide linked to to JunB

Several TRIM proteins are implicated in ubiquitination and act as single RING finger ubiquitin E3 ligases, which can affect the direct transfer of ubiquitin from E2~Ub/UBL to substrate proteins (Hong et al., 2008; Ishikawa et al., 2006; Tuoc and Stoykova, 2008).

Preliminary work could show a specific interaction between TRIM11 and the ULM FAT10 in a yeast two hybrid screen. Moreover, TRIM11 could interact in vivo with the FAT10 specific E2 enzyme USE1 and depletion of TRIM11 via siRNA knockdown significantly reduced the amount of FAT10 conjugates, indicating that TRIM11 may function as a FAT10 specific RING finger E3 ligase (A. Aichem, unpublished). Because enzyme sharing between different ULMs is not unusual it should be tested if TRIM11 not only possesses ubiquitin E3 ligase activity but is also able to cooperate with FAT10.

Results

99 Hence, we were interested, if the transcription factor JunB can covalently bind both, ubiquitin, as well as FAT10 and could be, beside USE1 (Aichem et al., 2010), p53 (Li et al., 2011) and huntingtin (Nagashima et al., 2011) a further substrate in the FAT10 conjugation pathway. If TRIM11 is either a ubiquitin or FAT10 specific E3 ligase with JunB as substrate, it would be expected that co-expression of these proteins would either enhance ubiquitin or FAT10 conjugation to JunB or lead to general augmentation of ubiquitin or FAT10 conjugate formation in transfected cells. Typically, E3 ligases confer substrate specificity in the ULM conjugation pathway but that doesn’t exclude that they are able to facilitate the modification of many different substrates.

We transiently transfected HEK293 cells with either a HA-tagged ubiquitin or HA-tagged FAT10 constructs alone, or together with a MYC-FLAG-tagged JunB and a HIS-tagged TRIM11 construct. After 24 h ectopic protein expression, ubiquitin and FAT10 were immunoprecipitated from cell lysates using anti-HA-agarose and expression was detected through immunoblotting with specific antibodies (Figure 21(a), (b) and (c)). β-actin served as a loading control for the prepared lysates. All samples were boiled in reducing gel sample buffer (10 % β-mercaptoethanol), to cleave non-covalent thioester-linkages, whereas isopeptide linkages remain stable.

(a)

100

Figure 21: Conjugate formation of JunB with either ubiquitin or FAT10 in vivo.

Co-immunoprecipitation of JunB-MYC-FLAG, HA-FAT10, HA-Ubiquitin and HIS-TRIM11 as indicated. HEK293 cells were transiently transfected with pCMV6-JunB-MYC-FLAG, pcDNA3.1-HA-FAT10, pCDNA-3.1-HA-Ubiquitin and pCDNA3-HIS/-A-TRIM11, or left untransfected. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). After immunoprecipitation against the HA-Tag of HA-FAT10 and HA-Ubiquitin 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 HA-FAT10 and Western blot analysis against the FLAG-tag of JunB (c). Arrow head indicates a putative homo/hetero-dimerized JunB-ubiquitin conjugate. β-actin served as a loading control.

(c) (b)

Results

101 Whole cell lysates were directly immunoblotted with an anti-HA antibody to reveal the expression of HA-tagged ubiquitin (~8 kDa) and HA-tagged FAT10 (~18 kDa) as depicted in Figure 21 (a). Further, cell lysates were directly immunoblotted with an anti-6HIS antibody to illustrate expression of HIS-TRIM11 (~52 kDa) and an anti-antibody, to detect FLAG-tagged JunB (~48 kDa) and JunB conjugates as depicted in Figure 21 (b) and (c).

All proteins could be detected at their predicted size. The amount of freely distributed ubiquitin was highest, when no further protein was co-expressed. The same result was observable for monomeric FAT10 (Figure 21 (a), input). Moreover, when ubiquitin was co-expressed with JunB or TRIM11, or with both proteins together, ubiquitin conjugates shifted to higher molecular weights (apparent in ubiquitin ladder, see Figure 21 (a), input).

Interestingly, immunoprecipitation against the HA-tag of ubiquitin and Western blot analysis through a HA–reactive antibody revealed, that ubiquitin-smear is almost absent in Figure 21 (a), lane 6, when ubiquitin is co-expressed together with JunB and TRIM11.

Strikingly, a prominent double band appears at the height of ~110 kDa. This size would equate to homo/hetero-dimerized JunB conjugated to ubiquitin (marked by arrow head), but this hypothesis indeed needs further investigation. Interestingly, this double band is also visible in Figure 21 (c), lane 3, when ubiquitin, co-expressed with JunB, is immuno-precipitated and analyzed with an anti-FLAG reactive antibody to detect ubiquitinated JunB.

Following immunoprecipitation of HA-Ubiquitin with anti-HA-agarose beads and Western blot analysis against the FLAG-tag of JunB, JunB could be on one hand detected for the most part at a size of 48 kDa, what would indicate a non-covalent interaction between JunB and ubiquitin (see Figure 21 (c) lane 3-5). The amount of co-immunoprecipitated JunB was clearly higher than the amount that unspecifically bound to the anti-HA-agarose beads apparent in Figure 21 (c) line 2, suggesting that the two proteins are mainly non-covalently linked (Figure 21 (c) lane 3). However, on the other hand a clear conjugate band at a height of ~56 kDa appears (Figure 21 (c), line 3), which size equates to mono-ubiquitinated JunB.

This conjugate linkage is not reducible what strongly argues for an isopeptide linkage.

Furthermore, a smear at higher molecular weights is visible, indicating that JunB becomes also poly-ubiquitinated. Conspicuous in this case is the prominent double band appearing at the height of ~110 kDa, as seen before (Figure 21 (a), lane 6, arrow head), which size equates to homo- or hetero -oligomerized JunB, conjugated to ubiquitin, but still remains to be investigated. Interestingly, co-expression of ubiquitin and JunB together with TRIM11 resulted in almost complete abrogation of Ub-JunB conjugate formation and TRIM11 over-expression led to the abolishment of ubiquitin smear (IP: Figure 21 (c) lane 4).

102 Surprisingly, after co-immunoprecipitation of HA-FAT10 and immunoblotting against the FLAG-tag of JunB, a high molecular weight (~66 kDA) conjugate double band of FAT10 and JunB was visible, although a JunB (48 kDa) remained partially non-covalently linked (Figure 21 (c) lane 5). The size of the double band fits to a monoFAT10ylated form of JunB which is not reducible and gives a strong hint that modification occurs covalently. Strikingly, the conjugate double-band disappeared almost completely when TRIM11 was co-expressed (Figure 21 (c) lane 6), similar to the previous case, when ubiquitin and JunB were co-expressed together with TRIM11. Generally, overexpression of TRIM11 led to a decreased protein level of ubiquitin, FAT10 and JunB (Figure 21 (a), (b), (c)), which could argue for accelerated protein degradation when TRIM11 is present in an abundant amount.

Collectively, these results show that JunB becomes ubiquitinated and FAT10ylated under the chosen conditions and provide evidence, that an interaction of JunB and ubiquitin or FAT10 occurs in vivo. So far, the putative FAT10 E3 ligase function of TRIM11 is not evidenced, and was investigated in an in vitro FAT10ylation assay at a later time point (see 6.2.13).

6.2.4 Proteasome inhibition augments conjugate formation between JunB and