Endoplasmic reticulum (ER) stress-induced, unfolded protein response (UPR)-mediated apoptosis is a protective mechanism whose failure contributes to malignant tumor progression. XAF1 is a stress-inducible, pro-apoptotic tumor suppressor which is inactivated in many human tumor cells. The molecular signal mechanism explaining its function in controlling ER stress, however, remains largely unclear. In this study, we explored the role of XAF1 in UPR signaling and ER stress-induced apoptosis.
XAF1 and GRP78 expression were determined by RT-PCR and immunoblot assay. the interplay of XAF1 with GRP78 was defined using immunoprecipitation and in vitro pull-down assays. ER stress was induced using thapsigargin, tunicamycin and brefeldin A. Flow cytometry was used to assess cell cycle progression and apoptosis.
XAF1 expression is upregulated at the transcriptional level in response to ER stress and this activation is abolished by suppression of PERK-Nrf2 but not of IRE1a or ATF6 signaling. This indicates that XAF1 is a target of Nrf2 in transcriptional level and activated by ER stress through the PERK pathway. This XAF1 induction greatly enhances the apoptotic response of tumor cells to ER stress while blockade of XAF1 induction reduces apoptotic sensitivity of tumor cells to ER stress. Intriguingly, XAF1 decreases the protein level of the ER stress sensor, GRP78, and consequently activates UPR signaling. Mechanistically, XAF1 binds directly to GRP78 and stimulates its proteasomal degradation in an E3 ligase ZNF313-dependent manner. XAF1 facilitates ZNF313 interaction with GRP78 and thereby promoting ZNF313-mediated K48 ubiquitination of GRP78. Unlikely wild-type XAF1, mutant XAF1 lacking GRP78- or ZNF313-binding domain shows no activity to promote ER stress-induced apoptosis.
Conclusion: XAF1 sensitizes cells to ER stress and drives apoptotic switch of UPR function away from cell cycle arrest by directly antagonizing GRP78 through the assembly of a ZNF313-mediated destruction complex. Our study identifies first a cell-fate decisions function of XAF1 under ER stress conditions.
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All authors have declared no conflicts of interest.