The proteasome is involved in Ambra1 degradation

Ambra1 has been described to be an indispensible regulator of autophagy initiation and is thereby involved in the process of cellular degradation (Di Bartolomeo et al., 2010; Fimia et al., 2007; Liang et al., 1999; Sun et al., 2009), but hitherto nothing is known about how Ambra1 itself becomes degraded and no data were available about the protein half life of Ambra1.

Results

137 To determine the protein half life of Ambra1 we performed cycloheximide chase experiments.

HEK293 cells were transiently transfected with a MYC-FLAG-tagged Ambra1 construct and cycloheximide (50 µg ml^-1) was added for the indicated time periods for 2.5 and 5 h or left untreated. Besides, cells were treated with or without MG132 (10 µM) for 6 h.

0 2.5 5 5 + 6 h MG132

0 50 100 150 200

CHX treatment [h]

relative Ambra1 expression

Figure 42: Determination of the turnover rate of ectopically expressed Ambra1

HEK293 cells were transiently transfected with a pCMV6-Ambra1-MYC-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. Whole cell lysates were subjected to Western blot analysis using a directly coupled horseradish peroxidase (HRP) -linked anti-FLAG mAb to evaluate Ambra1 expression. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). β-actin served as a loading control. One out of two experiments with similar outcome is shown. ECL signals of all experiments were quantified with Quantity One Software (BioRad). The ECL signal of Ambra1-MYC-FLAG transfected cells without cycloheximide treatment was set to unity. Mean values ± SEM of two independent experiments are shown.

The cycloheximide data illustrate, that the protein turnover of Ambra1 only slightly decrease to ~80 % after 5 h of cycloheximide treatment. Surprisingly, MG132 treatment led to a considerable accumulation of Ambra1 to 160 %, which clearly indicate an involvement of the proteasome in Ambra1 degradation.

We next aimed to determine the protein turnover rate of Ambra1 in presence of FAT10.

HEK293 cells were transiently transfected with a HA-tagged FAT10 and MYC-FLAG-tagged Ambra1 construct. Cycloheximide (50 µg ml^-1) was added for the indicated time periods for 2.5 and 5 h or left untreated. Moreover, cells were treated with or without MG132 (10 µM) for 6 h. Whole cell lysates from transfected cells were analyzed for expression of the respective proteins with a FLAG-reactive antibody, to detect MYC-FLAG tagged Ambra1 and an anti-HA antibody to reveal HA-FAT10 expression.

138

Figure 43: Determination of Ambra1 turnover rate in the presence of FAT10

HEK293 cells were transiently transfected with (a) pCMV6-Ambra1-MYC-FLAG and pcDNA3.1-HA-FAT10 plasmids. 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. Whole cell lysates were subjected to Western analysis using a directly coupled horseradish peroxidase (HRP) -linked anti-FLAG mAb to evaluate Ambra1 expression and an HA-reactive antibody to evaluate FAT10 expression. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). β-actin served as a loading control. One out of two experiments with similar outcome is shown. The ECL signal of Ambra1-MYC-FLAG and pcDNA3.1-HA-FAT10 transfected cells without cycloheximide treatment was set to unity. Mean values ± SEM of two independent experiments is shown.

Surprisingly, Ambra1 turnover in presence of FAT10 illustrates a faster degradation progress than in absence of FAT10 (see Figure 42). The half life of Ambra1 in presence of FAT10 is about 2.5 h and interestingly, almost no rescue with MG132 could be observed (see Figure 43). These results suggest, that co-expression of FAT10 alters the degradation rate of Ambra1 and interaction leads presumably to another degradation machinery than the proteasome. In contrast, the FAT10 half life in presence of Ambra1 was calculated to be approximately 1 h and MG132 treatment led to an increased accumulation of monomeric FAT10. These results indicate that FAT10 co-expression leads to accelerated Ambra1 protein turnover, whereby the turnover rate of monomeric FAT10 is hardly altered, when Ambra1 is co-expressed.

Next, we aimed to determine the Ambra1 turnover in presence of FAT10 and TRIM11.

HEK293 cells were transiently transfected with an HA-tagged FAT10, a MYC-FLAG-tagged Ambra1, together with a HIS-tagged TRIM11 construct. Cycloheximide (50 µg ml^-1) was added for the indicated time periods for 2.5 and 5 h or left untreated. Further, cells were treated with or without MG132 (10 µM) for 6 h.

Results

139 Whole cell lysates from transfected cells were analyzed for expression of the respective proteins with a FLAG-reactive antibody, to detect MYC-FLAG tagged Ambra1 (Figure 44 (a)), anti-HA antibody to reveal HA-FAT10 expression (Figure 44 (b)) and an anti-6HIS-Pox antibody to visualize HIS-TRIM11 expression (Figure 44 (c)).

Figure 44: Determination of the turnover rate of ectopically expressed Ambra1 in the presence of FAT10 and TRIM11

HEK293 cells were transiently transfected with pCMV6-Ambra1-MYC-FLAG, pcDNA3.1-HA-FAT10, together with pcDNA3-HIS/-A-TRIM11. Before cell lysis, cells were treated with the proteasome inhibitor MG132 (10 µM) for 6 hours and cycloheximide (50 µg ml^-1) was added for different time periods, as indicated. Whole cell lysates were subjected to Western blot analysis using a directly coupled horseradish peroxidase (HRP) -linked anti-FLAG mAb to evaluate Ambra1 expression, an HA-reactive antibody to evaluate FAT10 expression and a 6HIS-POX antibody to determine HIS-TRIM11 expression. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). β-actin served as a loading control. One out of two experiments with similar outcome is shown.

The ECL signal of Ambra1-MYC-FLAG, pcDNA3.1-HA-FAT10 and pcDNA3-HIS/-A-TRIM11 transfected cells without cycloheximide treatment was set to unity. Mean values ± SEM of two independent experiments is shown.

(a)

(b)

(c)

140 The cycloheximide data illustrate, that the protein turnover Ambra1 in presence of FAT10 and TRIM11 is similar compared to the degradation rate when Ambra1 alone was ectopically expressed in the cell (see Figure 42). 5 h after cycloheximide addition the protein level decreased to ~80% and strikingly, MG132 treatment led to an enhancement of Ambra1 to 140 % (see Figure 44 (a)), which again clearly indicate an involvement of the proteasome in Ambra1 degradation, in contrast to Ambra1 when co-expressed together with FAT10, where no rescue of Ambra1 after MG132 treatment could be observed (see Figure 43, lane 4). The role of TRIM11 in this process is currently under investigation and needs to be clarified.

These data let strongly suggest, that Ambra1 becomes degraded through different pathways, whereby the proteasome is presumably one of the degradation machineries engaged in Ambra1 depletion.