Cul5-ASB11 E3 complex mediates BIK ubiquitination
BIK is a labile protein, but its abundance can be drastically accumulated and stability can be greatly enhanced after treatment of cells with proteasome inhibitor bortezomib or MG132 [124]. This implies an important role of ubiquitin-proteasome system in BIK proteolysis. However, ubiquitin E3 ligase that can catalyze BIK ubiquitination is still unknown. Previous study in our lab identified Cul5-ASB11 E3 complex as a candidate ubiquitin ligase responsible for BIK ubiquitination, based on the fact that BIK ubiquitination was enhanced by ASB11 overexpression in 293T cells (Fei-Yun Chen, unpublished results).
Thus, we purposed a hypothesis that BIK is a direct substrate of Cul5-ASB11 E3 complex. To test our hypothesis, we first examined the interaction between BIK and ASB11 by employing a co-immunoprecipitation (co-IP) analysis. In this experiment, Flag-ASB11 was transfected into 293T cells, and cell lysate was used to perform the co-IP with anti-Flag M2 beads. We found that endogenous BIK was specifically coprecipitated with Flag-ASB11 (Fig. 1), which supports our hypothesis. Next, the interaction between ASB11 and BIK was confirm by the in vitro binding assay, in which the purified Myc-ASB11 was immobilized on anti-Myc beads and co-incubated with 3xFlag-BIK purified from transfected cells (Fig. 2). Thus, the in vitro and in vivo interaction assays indicate BIK as a binding partner of ASB11.
To further confirm that BIK is a direct substrate of ASB11-based ubiquitin ligase, we performed an in vitro ubiquitination assay. The ROC2/Cul5/Elongin B/C/
ASB11 E3 ligase complex purified from cotransfected cells was capable of promoting
polyubiquitination of BIK in an E1/E2 dependent manner (Fig. 3). Together, our results indicate that BIK is a substrate of this E3 ligase complex.
ASB11 facilitates BIK degradation through ubiquitin-proteasome pathway
Following the discovery of ASB11-mediated BIK ubiquitination, we turned our interest into the biochemical and physiological significant of this ubiquitination. To explore the role of ASB11 in BIK regulation, we transfected Flag-ASB11 into 293T cells and then investigated its influence on BIK homeostasis. We found that the cellular abundance of BIK protein was reduced by the overexpression of ASB11 (Fig. 4).
Furthermore, this ASB11-promoted BIK downregulation was blocked by treatment of cells with proteasome inhibitor MG132, indicated the participation of proteasome system in BIK downregulation (Fig. 4).
Next, we employed the cycloheximide (CHX) assay, in which cells were treated with the protein synthesis inhibitor CHX to monitor the half-life of proteins. The CHX assay demonstrated a decreased stability of BIK protein in ASB11 overexpressed 293T cells (Fig. 5). Together, our results indicate that ASB11 promotes BIK degradation through ubiquitin-proteasome pathway.
ASB11 promotes cell survival and antagonize the DNA damage-induced cell death
Based on the fact that BIK is induced by the DNA damage response to promote apoptosis, we reasoned that overexpression of ASB11 might be able to antagonize the DNA damage-induced cell death. To verify this notion, we analyzed the influence of ASB11 overexpression on the sensitivity of cancer cells to DNA damage agents. We established two HCT116 cell lines that stably expressed wild-type and SOCS box truncated ASB11 mutant, which cannot form Cul5 ubiquitin ligase complex. The cells
were treated with DNA damage agent doxorubicin, etoposide, or cisplatin for 48 hr followed by applying PI (propidium iodide) stain and flow-cytometry to monitor the sub-G1 (apoptotic) cells. We found that ASB11 overexpression induced a pro-survival effect as it partially inhibited the DNA damage-induced apoptosis (Fig. 6). In contrast, the overexpression of SOCS box truncated ASB11 did not promote cell survival (Fig.
6), indicating that the pro-survival function of ASB11 is dependent on the formation of Cul5-ASB11 complex. Together, our results suggest that ASB11 promotes cell survival and antagonizes the DNA damage agents induced apoptosis by facilitating the degradation of pro-apoptotic BIK protein through the ubiquitin-proteasome pathway.
ASB11 is transcriptionally repressed by DNA damage in a p53-dependent manner
After demonstrating a blockage effect of ASB11 on DNA damage-induced cell death, we wanted to investigate whether ASB11 could be regulated in response to DNA damage. To this end, we treated HCT116 cells with DNA damage agent doxorubicin, 5-fluorouracil, or cisplatin for 24 hr and found that each of these agents led to a dose-dependent downregulation of ASB11 mRNA level (Fig. 7). Since p53 is a key player of the DNA damage response, we next examined the contribution of p53 pathway to DNA damage-induced ASB11 downregulation. Remarkably, in p53-deficient HCT116 cells, ASB11 mRNA levels were unchanged or even slightly elevated in response to DNA damage agents (Fig. 7). These findings indicate that DNA damage represses ASB11 mRNA through a p53-dependent mechanism.
To further evaluate the capability of p53 in regulating ASB11 mRNA, we transfected p53-null H1299 cells with p53 and found that this ectopic p53 induced a decrease of ASB11 mRNA even without DNA damage. Likewise, p53-proficient
HCT116 cells expressed a lower level of ASB11 mRNA than that in p53-deficient HCT116 cells (Fig. 8). These findings indicate that ASB11 is transcriptionally suppressed by p53.
DNA damage prevents BIK from ASB11-mediated ubiquitin-proteasomal degradation in a p53 dependent manner
Consistent with the DNA damage-induced ASB11 downregulation and ASB11-induced BIK degradation, BIK protein level was elevated in HCT116 cells in response to DNA damage agent doxorubicin, 5-fluorouracil, or cisplatin. However, this elevation was greatly impaired in p53-deficient HCT116 cells (Fig. 9). By treating cells with proteasome inhibitor MG132, we showed that DNA damage-induced BIK elevation was mainly due to an inhibition of its proteaosmal degradation, as MG132 greatly increased BIK level in cells without receiving DNA damage agents but not in damaged cells (Fig.
10a-c, right panels). This difference in BIK proteasomal degradation seen in the damaged and undamaged conditions was greatly diminished in p53-deficient HCT116 cells (Fig. 10a-c, left panels). Furthermore, in vivo ubiquitination assay showed that DNA damage agent doxorubicin and 5-fluorouracil decreased the ubiquitination level of BIK in p53 proficient HCT116 cells but not in p53 deficient HCT116 cells (Fig. 11).
Together, these data support an idea that DNA damage acts through p53 to downregulate ASB11, thereby preventing BIK ubiquitination and degradation.
To strengthen this idea, we further tested the contribution of ASB11 to DNA damage-induced BIK stabilization. CHX assay demonstrated an increased BIK half-life by cisplatin treatment. However, in ASB11 knockdown cells, such BIK stabilization is largely compromised (Fig. 12). Taken together, we provide a novel model in which the
DNA damage-induced p53 pathway transcriptionally suppresses ASB11 to inhibit ASB11-mediated ubiquitination and proteasomal degradation of BIK protein.
ER stress transcriptionally upregulates ASB11 expression and promoted BIK degradation in the XBP1 dependent pathway
Having discovered a role of ASB11 in DNA damage-induced BIK stabilization, we wondered whether ASB11-mediated BIK ubiquitination can also be regulated under other stressed conditions. Through literature search, we found that both ASB11 and BIK are ER resident proteins [78, 125]. Furthermore, BIK is involved in calcium homeostasis function of ER [126] and is regulated by ER stress sensor GRP78 [68].
Based on these findings, we decided to explore a linkage of ER stress to ASB11-mediated BIK ubiquitination..
We used ER stress inducer thapsigargin and tunicamycin, which disturbs protein homeostasis in ER by alternating ER calcium homeostasis and blocks protein N-glycosylation, respectively, to activate the UPR. After 24 hours treatment with thapsigargin and tunicamycin, the mRNA level of ASB11 was significantly upregulated in the 293T cells (Fig. 13). Furthermore, knockdown of XBP1 blocked the induction of ASB11 expression, indicating that ER stress response promotes ASB11 expression through the IRE1-XBP1 pathway (Fig. 13). In line with this finding, the BIK protein abundance was decreased by tunicamycin treatment in 293T cells (Fig. 14).
Furthermore, this BIK downregulation was abolished by MG132 treatment (Fig. 14), indicating that ER-stress response promotes BIK degradation through the ubiquitin-proteasome system. We further showed that ER-stress inducers failed to downregulate BIK protein abundance in the XBP1 knockdown 293T cells (Fig. 15). Thus, our
findings indicate that ER stress acts through IRE1-XBP1 pathway to induce ASB11 expression, thereby promoting BIK degradation.