Identification of JunB in mass spectrometry analysis

6.2.15 Identification of JunB in mass spectrometry analysis

To further ensure that the conjugate bands consist of JunB and FAT10 and in order to identify, if FAT10ylated JunB contains post-translational modifications, mass spectrometry analysis was performed. Moreover, we were interested if the lysine involved in conjugate formation, could be confirmed.

Therefore, HEK293 cells were transiently transfected either with pcDNA3.1-HA-FAT10 and pCMV6-JunB-MYC-FLAG or left untransfected. After 24 h of ectopic protein expression, JunB was immunoprecipitated from cell lysates using anti-FLAG-agarose. Samples were boiled in reducing sample buffer containing 10 % β-mercaptoethanol and proteins were separated on 4-12 % Bis-Tris-gels. The gel was stained for 10 minutes with Coomassie (see Table 9) and destained for 30 min with destaining solution (see Table 10) and the two bands appearing at the height 66 kDa which equate to JunB-FAT10 conjugates (Figure 35 (a), indicated as (2) and (3)) were cut out and analyzed by mass spectrometry. As a negative control, gel pieces were cut out from the untransfected control lane (Figure 35 (a), lane 1) at the same height where conjugate bands are appearing (Figure 35 (a) indicated as 1.1 and 1.2) and sent to mass spectrometry analysis, as well.

As a further control, samples were analyzed by Western blotting and probed with a anti-HA-reactive antibody, to ensure that the bands, appearing at the expected height in the Coomassie stained gel, coincide with the JunB-FAT10 conjugate bands detected in the Western blot (Figure 35 (b)).

Results

123

Figure 35: Mass spectrometric analysis of the JunB-FAT10 conjugate

HEK293 cells were co-transfected with 6 µg pCMV6-JunB-MYC-FLAG and 6 µg pcDNA3.1-HA-FAT10 expression plasmids, respectively or left un-transfected. After immunoprecipitation against the FLAG-Tag of JunB, samples were boiled in gel sample buffer containing 10% β-mercaptoethanol (reducing conditions) and proteins were separated on 4-12% Bis-Tris SDS gels. They were either stained with Coomassie a) or subjected to Western blot analysis using a HA-reactive antibody conjugated to horseradish peroxidase to detect conjugate formation (b).

The asterisks mark the signal corresponding to antibody light or heavy chains used for immunoprecipitation.

Double bands appearing (2,3) in the Coomassie gel which equates to FAT10ylated JunB at the height of ~66 kDa were excised and analyzed by mass spectrometry. As negative control bands at the same height ~66 kDa of untransfected HEK293 cells were cut out (1.1; 1.2) as well and analyzed by mass spectrometry.

Mass spectrometric analysis (Thermo LTQ Orbitrap, Proteomics facility at the University of Constance, A. Marquart) revealed that the lower (Figure 35 (2)) as well as the upper conjugate band (Figure 35 (3)) contained JunB peptide fragments. However, detailed analysis of the lower band (2) revealed, that two JunB-peptide fragments could be detected, in comparison to the upper band (3), where three peptide fragments where measured, suggesting a post-translational modification for the latter case (for mass spectrometry results see addendum, chapter 10. Unfortunately no FAT10 could be identified in the analyzed samples and therefore sent to H. Urlaub group (MPI Göttingen) for further analysis by a program called Chop'N'spice, a mass spectrometric approach that has been described to allow the identification of endogenous SUMO conjugated peptides (Hsiao et al., 2009). No JunB or FAT10 was detected in the untransfected control bands, which served as negative control (1.1 and 1.2).

b) a)

124 6.2.16 Post-translational modification: Phosphorylation of JunB on Serine 259?

Our previous studies demonstrated that JunB becomes stably attached to FAT10 (see chapter 6.2.3, 6.2.4 and 6.2.7), and interestingly, conjugate formation resulted in a double band appearing at the height of approximately 66 kDa. To address the issue, whether a post-translationally modified form of JunB becomes conjugated to FAT10, we considered it to be likely, that phosphorylation could be the reason.

Figure 36: Serine259 phophorylated form of JunB becomes conjugated to FAT10

HEK293 were single transfected with MYC-FLAG-tagged JunB or HA-tagged FAT10 or double-transfected. After 24 h, cells were lysed and immunoprecipitated against the HA-tag of FAT10. Samples were boiled in gel sample buffer containing 10% β-mercaptoethanol (reducing conditions) and proteins were separated on 4-12% Bis-Tris SDS gels and subjected to Western blot analysis using a (a) polyclonal JunB antibody (ab31421) or (b) serine 259 (S259) phospho-specific JunB antibody (ab30628) to evaluate JunB expression, or (c) a HA-reactive antibody to detect FAT10 expression. β-actin served as a loading control. Arrow heads indicate JunB-FAT10 conjugates.

Asterisks mark unspecific bands.

HEK293 were transiently transfected with a MYC-FLAG-tagged JunB, a HA-tagged FAT10 or double-transfected. 24 h after ectopic protein expression, samples were immunoprecipitated with HA-agarose against the HA-tag of FAT10 to accumulate FAT10 and FAT10 conjugates and samples were immunoblotted with a polyclonal JunB antibody (ab31421) (Figure 36 (a)), or a serine 259 (S259) phospho-specific JunB antibody (ab30628) (Figure 36 (b)) to detect JunB, and a HA-reactive antibody (Figure 36 (c)) to visualize FAT10 expression.

(a)

(b)

(c)

Results

125 Interestingly, co-expression of JunB together with FAT10 resulted in increased JunB expression, which was detectable in the JunB-blot ((Figure 36 (a), input) as well as in the phospho-JunB(S259) blot (Figure 36 (b), input), similar to the case, when FAT10 was induced with proinflammatory cytokine INF-γ and TNF-α (see 6.2.12). In comparison, FAT10 expression decreased significantly when co-expressed together with JunB (Figure 36 (c), input). The fact, that the highest and coincidentally the most prominent JunB band is recognized by both, the polyclonal JunB and the Ser259 phospho-specific JunB antibody suggested, that this band presents a phosphorylated form of JunB.

Analysis of co-immunoprecipitated JunB and immunoblotting with either the polyclonal JunB, or phospho-Ser259 specific JunB antibody illustrate, that a JunB-FAT10 conjugate is formed apparent in a high molecular weight double band at 66 kDa. Interestingly, this double band was not detectable, when samples were analyzed by immunoblotting with a Ser79 phospho-specific antibody (data not shown), suggesting a phospho-specific interaction of FAT10 with a Ser259 phosphorylated form of JunB.

6.2.17 FAT10 and JunB co-localize at the nuclear membrane and in the cytosol