The fusion oncoprotein PML-RAR induces endoplasmic reticulum-associated degradation of N-CoR and ER stress

PML-RAR a , a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor- a (RAR a ), causes acute promyelocytic leukemias (APL). Although the role of nuclear PML-RAR a has been extensively studied, a significant amount of PML-RAR a is in the cytoplasm. The role cytoplasmic PML-RAR a plays in leukemogenesis is unknown. Here we report that PML-RAR a induces the N-CoR accumulation in the endoplasmic reticulum (ER), leading to the induction of ER stress and the processing of activating transcription factor 6 (ATF6), the unfolded protein response. PML-RAR a stimulates the ubiquitylation of N-CoR via Ubc6 that is involved in the protein quality control. This ER-associated degradation (ERAD) of N-CoR reduces the soluble N-CoR protein levels in the nucleus. The two N-CoR-interacting sites in PML-RAR a are required for the ERAD of N-CoR, suggesting the aberrant binding of PML-RAR a to N-CoR may induce the ERAD of N-CoR. Over-expression of N-CoR induces the differentiation of APL-derived NB4 cells, suggesting that the low levels of N-CoR in the nucleus may contribute at least partly to PML-RAR a -mediated leukemogenesis.


INTRODUCTION
Khan, M. M. et al. 6 finger domain of PML were substituted with serine. In PML-RAR∆C-C, the whole coiled-coil domain (residues 228 to 365) was deleted. Similarly, in the B boxes deletion mutant, the deleted region was the two B-boxes and intervening region, extending from residue 130 to 227. In the PML-RARα (AHT) mutant construct, the A, H and T residues at 223, 224 and 227 sites, which are required for interaction with N-CoR molecule, were replaced by G, G and A residues respectively. The HA-ATF6 expression vector (pCGN-ATF6) was described previously (22). To express Ubc5 or Ubc6 in 293T cells, pcDNA3.1 vector (Invitrogen) was used. In Ubc5(CS) and Ubc6(CS) expression vectors, the active cysteine residue was replaced with serine.
Immunocytochemistry ---293T cells were co-transfected with pact-N-CoR-Flag GST pull-down assays and co-immunoprecipitation ---GST pull-down assays were performed essentially as described (8). The GST fusion of full-length PML-RARα or various domains of PML were used with various in vitro-translated Ubc enzymes in a binding buffer containing 10 mM HEPES (pH 6.5), 2.5 mM MgCl 2 , 50 µM ZnCl 2 , 0.5 mM DTT, and 0.01% BSA. For co-immunoprecipitation of PML-RARα and Ubc6, lysates from 293T cells transfected with 3 µg pact-HA-PML-RARα and 3 µg of FLAG-Ubc6 expression vector were prepared by sonication in LSLD buffer (50 mM Hepes, pH 6.5, 50 mM NaCl, 20 µM NaF, 20% glycerol, and a protease inhibitor cocktail). Immunoprecipitation was performed with anti-Flag antibody, and the anti-HA Khan,M. M. et al. 11 antibody was used for Western blotting. For co-immunoprecipitation between endogenous Ubc6 and PML-RARα, lysate of NB4 cells prepared in above mentioned buffer was immunoprecipitated with UbcH6 antibody (Boston Bio-chemicals) and Western was done with anti-RARα antibody to detect endogenous PML-RARα protein.
For co-immunoprecipitation of PML-RARα and PDI, NB4 cells were lysed in PBS-0.1% NP40 by mild sonication, immunoprecipitated with the anti-PDI antibody and blotted with the anti-RAR antibody (Santa Cruz). To detect the association of N-CoR with PDI, a cytosolic fraction of NB4 cells was prepared as described below, immunoprecipitated with anti-PDI antibody, washed with PBS, and N-CoR was detected with goat anti-N-CoR antibody.
Flow cytometry ---The retroviral expression plasmid for N-CoR was constructed using the MSCV (murine stem cell virus)-based retroviral vector. The plasmid pMSCV-N-CoR encodes both N-CoR and GFP separated by internal ribosome entry site so that they are expressed together. NB4 cells (10 x 10 6 cells) were transfected with 40 µg of pMSCV-N-CoR or MSCV empty vector using DMRIE-C reagent (Invitrogen) for 24 hr, and then complete medium was added and the cells were cultured for an additional 48 hr. After that, the cells were collected, washed twice with PBS-0.5% BSA, and incubated in 500 µl PBS-0.5% BSA with RPE-conjugated monoclonal mouse anti-human CD14 antibodies (DAKO) for 60 min on ice. The cells were then washed with PBS-0.5% BSA and analyzed on a FACScan flow cyometer (Becton Dickinson). The GFP-positive cells comprised 3-4% of all the NB4 cells. To detect the exogenous N-CoR in NB4 cells, lysate prepared from nuclear fraction of transfected 12 NB4 cells was subjected to Western blotting using anti-Flag antibody.
To investigate the effect of tunicamycin on RA-mediated differentiation of NB4 cells, NB4 cells were treated with RA (1 µM) alone or with RA and tunicamycin (2.5 µg/ml). Tunicamycin was added in RA-treated cells 24 hours before harvesting.
Cells were incubated with RPE-conjugated monoclonal mouse anti-human CD11b antibody (DAKO) and FACS analysis was done as described above.

RESULTS
The PML-RAR /N-CoR complex is recruited to the ER ---We previously demonstrated that PML-RARα has two sites that directly bind to N-CoR (8,9). It is possible that when the N-CoR molecule binds to PML-RARα through these two sites, it adopts an aberrant protein conformation. This may cause N-CoR to become a target of the protein quality control machinery located in the ER. To test this, we assessed whether the PML-RARα/N-CoR complex is bound by protein disulphide isomerase (PDI) and BiP/GRP78 (immunoglobulin binding protein/glucose-regulated protein), two of the ER resident protein, as they have been shown to mediate ER retention and degradation of poorly assembled and mis-folded proteins (25). 293T cells were cotransfected with the PML-RARα and N-CoR expression vectors and stained with antibodies against either protein together with antibodies that recognize the endogenous PDI and BiP molecule, followed by confocal microscopy analysis. When N-CoR or PML-RARα was expressed alone, the N-CoR signals were localized in the nuclear dotlike structures known as NBs, while PML-RARα was found predominantly in the cytosol like the endogenous PDI and BiP (Fig. 1A). As reported (6) 14 with antibodies specific to marker protein of the compartment. Fractions 10-16 were designated ER by their possessing of either PDI or BiP, and fractions 4-8 were designated Golgi because they contained Golgin-97, the integral membrane protein localized on the cytoplasmic face of the Golgi apparatus (26). N-CoR was found predominantly in the ER fraction and to some extent, also in the Golgi fraction. PML-RARα was known to form a dimer or oligomer (27,28), and we could detect both monomer and dimer under standard SDS-PAGE condition, probably due to very strong capacity to form a dimer. Both the dimeric and monomeric forms of PML-RARα were found in the ER fraction, while the monomeric PML-RARα was also abundant in the Golgi fraction. Since it was recently shown that the ER-Golgi traffic is a prerequisite for efficient ER degradation of mis-folded proteins (29), the localization of N-CoR and PML-RARα in the ER and Golgi is consistent with our notion that the N-CoR protein complexed with PML-RARα has an aberrant protein conformation.
When APL-derived NB4 cells were assessed for endogenous PML-RARα and PDI signals, significant overlapping between them was observed ( Fig In the Nycodenz fractionation of the cytoplasmic lysates of NB4 cells, N-CoR was predominantly recovered in the Golgi fraction, though the ER fraction also contained some N-CoR protein (Fig. 2C). The monomeric form PML-RARα was found in the ER fraction, while those of dimeric form were recovered in the Golgi fraction. The difference in the distribution of PML-RARα and N-CoR between transfected 293T cells and NB4 cells may be due to the relatively high amounts of the PML-RARα/N-CoR proteins in the transfected 293T cells. These observations, together with our previous finding that PML-RARα directly associates with N-CoR (8,9), suggest that N-CoR bound to PML-RARα is recruited to the ER.

PML-RAR /N-CoR targeted to the ER triggers ER stress ---We previously
shown that N-CoR directly binds to PML-RARα via the two sites (8). To examine whether the N-CoR protein bound to PML-RARα has the different conformation compared to the unbound form, we investigated the proteases sensitivity of N-CoR.
Whole-cell lysates were prepared from the RA-treated and untreated NB4 cells, and digested with different concentration of protease K or trypsin (Fig. 3A). The results of Western blotting indicated that the pattern of N-CoR fragments obtained by both proteases were different between the RA-treated and untreated cell lysates. In the RAtreated lysates, the N-CoR protein appeared to be more sensitive to both proteases.
These results suggest that the N-CoR protein conformation changes by binding to PML-

RARα.
Since PDI has been shown to mediate ER retention and degradation of poorly Khan, M. M. et al. 16 assembled and mis-folded proteins (25), we tried to confirm the association of PDI with PML-RARα and N-CoR by a co-immunoprecipitation assay using lysates prepared from NB4 cells. Significant amounts of PML-RARα and N-CoR proteins, but not another corepressor mSin3A, were co-precipitated by the anti-PDI antibody, confirming that N-CoR bound to PML-RARα forms a complex with PDI in NB4 cells (Fig. 3B).
Thus, the PML-RARα/N-CoR complex is recruited to the ER through its interaction with the ER resident protein PDI.
ATF6, a bZIP transcription factor, is synthesized as an ER-localized transmembrane protein with a lumenal-sensing domain and a cytosolic transcription transactivation domain. Upon ER stress, ATF6 translocates from the ER to the Golgi where it is processed to its active form and is then transported to the nucleus, where it binds to the ER stress-inducible elements of a group of chaperone genes (22,30). To investigate whether the PML-RARα/N-CoR complex induces ER stress and therefore activates the processing of ATF-6, we studied the status of the ATF6 protein in NB4 cells by Western blotting the cell lysates with anti-ATF6 antibody (Fig. 3C). A processed form of ATF6 [pATF6(N)] was detected in NB4 cells, indicating that NB4 cells are in a state of elevated ER stress. When NB4 cells were treated with RA, the amount of processed ATF6 decreased considerably, indicating that RA treatment relieves the stress, possibly through the RA-mediated degradation of the PML-RARα protein (31). In addition, HeLa cells cotransfected with the plasmids expressing N-CoR, PML-RARα and ATF6 also contained the cleavage product and deglycosylated form of ATF6 (Fig. 3D). This form was not detected when ATF6 was expressed on its own. Localization of high amounts of N-CoR/PML-RARα in the Golgi may disturb the glycosylation of ATF6 in the ER, leading to the accumulation of the deglycosylated form of ATF6. We performed this latter experiment in HeLa cells because they have been frequently used in the study of the ER stress-induced activation of ATF6. In the control experiments, HeLa cells transfected with ATF6 alone were treated with tunicamycin or DTT. As reported previously (30), these treatments also generated the cleavage product and deglycosylated form of ATF6. Thus, the PML-RARα/N-CoR complex is recruited to the ER lumen and induces ER stress and the UPR.  (33), whereas its half-life was reduced to about 1 hr by coexpression of PML-RARα (Fig. 4C). In contrast, the half-life of the mixture of soluble and insoluble forms of N-CoR (in the RIPA buffer lysates) was found to be prolonged about two-fold by the presence of PML-RARα, from 6 hours in the absence of PML-RARα to 12 hours in its presence (Fig. 4D). These findings suggest that PML-RARα causes the N-CoR protein to rapidly accumulate in the ER as the insoluble form, possibly by blocking its physiological turnover and diverting the aggregated N-CoR to the ERAD-mediated degradation pathway.

PML-RAR induces insoluble N-CoR protein accumulation and causes the reduction of soluble N-CoR in the nucleus
It has been reported that poorly assembled and mis-folded secretory proteins are ubiquitinated and degraded by the ERAD-mediated protein quality control mechanism (19,20,21). To investigate whether PML-RARα-mediated insolubility of N-CoR involves a similar mechanism, we performed in vivo ubiquitination assays using  tail to the ER and is involved in the ER-mediated protein quality control mechanism (35,36). The association between Ubc6 and PML-RARα was further confirmed by coimmunoprecipitation and immunocytochemistry assays using 293T cells transfected with the Ubc6 and PML-RARα expression plasmids. The HA-tagged PML-RARα was precipitated along with the Flag-linked Ubc6 protein, indicating that ectopically expressed PML-RARα and Ubc6 form a complex in vivo (Fig. 5B, left panel).
Similarly, UbcH6 antibody precipitated endogenous PML-RARα from the lysate of NB4 cells (Fig. 5B, right panel). Expression of UbcH6 protein in NB4 cells was confirmed by Western blotting using the above mentioned anti UbcH6 antibody (data not shown). Moreover, ectopically expressed Ubc6 was found to colocalize with exogenous PML-RARα and endogenous PDI in the cytosol of 293T cells (Fig. 5C). To identify the domain of PML-RARα that interacts with Ubc6, we used the GST fusion proteins containing various portions of PML (Fig. 5D). The GST fusion proteins that contained the Ring finger domain or the B box bound efficiently to the in vitrotranslated Ubc6 protein.
We confirmed that Ubc6, in the presence of PML-RARα, specifically mediates N-CoR ubiquitination by using a dominant negative form of Ubc6. All E2 enzymes contain an active cysteine residue that is used for ubiquitin-thiolester formation. In the case of Ubc2 (RAD6) and UbcH10, alteration of this conserved cysteine to serine causes theses proteins to form a ubiquitin ester that cannot be transferred to the target proteins, thereby generating the dominant negative forms of these proteins (37,38). We examined the effect of similar mutants of Ubc6 and Ubc5 (CS mutants) on N-CoR ubiquitination in MG132-treated 293T cells that ectopically express N-CoR-Flag and myc-ubiquitin with or without PML-RARα. The Ubc6 mutant inhibited the ubiquitination of N-CoR in a dose-dependent manner, whereas the Ubc5 mutant had no effect (Fig. 5E). These findings indicate that Ubc6 is specifically involved in the PML-RARα-mediated ubiquitination of N-CoR and may also play a role in its anchorage to the ER as Ubc6 is an integral membrane protein that is anchored to the ER (37,38).  (8). We speculated that this aberrant binding may induce an abnormal conformation of N-CoR, and that this leads to ER stress and ERAD.

The roles played by each PML-RAR domain in targeting N-CoR to the ER
Consistent with this notion, we found that the deletion of the coiled-coil region (∆C-C) or point mutations in the CoR box (AHT) abrogated the capacity of PML-RARα to induce N-CoR insolubility (Fig. 6A, left panel). Consistent with these effects on N-CoR insolubility, both of these mutants (∆C-C and AHT) were also defective in enhancing the ubiquitination of N-CoR (Fig. 6A, right panel). Surprisingly, however, while the Ring finger mutant (RF) induced N-CoR ubiquitination at the same level as wild-type PML-RARα (Fig. 6A, right panel), it failed to induce any N-CoR protein insolubility (Fig. 6A, left panel). PML did not stimulate insolubility or ubiquitination of N-CoR when over-expressed in 293T cells with N-CoR (Fig. 6A, both panels).
To further characterize the roles the individual PML-RARα domains play in the insolubility of N-CoR, we investigated the subcellular distribution of ectopically expressed N-CoR in 293T cells together with various PML-RARα mutants (Fig. 6B).
When expressed alone, N-CoR was uniformly distributed in the nucleus and formed a micro-speckled pattern (Fig. 1A). When N-CoR was co-expressed with PML, N-CoR was recruited into nuclear dot-like structures where it co-localized with PML (Fig. 6B).  repression as expected (Fig. 7B). The Ubc5(CS) mutant did not affect the Mad-mediated repression. Thus Ubc6 is required to maintain the activity of typical tumor suppressor Mad by reducing the levels of the N-CoR protein complexed with PML-RAR.
We also investigated the possible contributory role of ER stress in the transformation of NB4 cells by examining the effect of tunicamycin on the RAmediated differentiation of NB4 cells. As shown above (Fig. 3D), tunicamycin induces the ER stress. When it was employed with RA, it blocked the RA-mediated differentiation of NB4 cells significantly as marked by down-regulation of monocytic differentiation marker CD11b (Fig. 7C). In addition, we observed the tunicamycin treatment also blocked the morphological differentiation of NB4 cells into the macrophage-like shapes (data not shown). These results suggest the possible role of ER stress in the PML-RARα-induced leukemogenesis.

DISCUSSION
Based on our findings, we hypothesize that the cytoplasmic PML-RARα molecule engages the N-CoR molecule just after translation in the cytosol by binding to it with its PML-derived coiled-coil domain and its N-CoR-derived CoR motif. The resulting complex probably acquires an abnormal conformation due to this aberrant binding and become a target of the protein quality control machinery located in the ER.
We observed that various normal protein, including the transcription factors c-Myb and ATF-2, were not localized in the ER when they were overexpressed (Khan and Ishii, unpublished results), supporting the notion that an abnormal conformation of the protein is critical for the ER retention. When PML-RARα alone was overexpressed in 293T cells, PML-RARα is located in the ER (Fig. 1A), suggesting that this fusion protein itself has an abnormal conformation. It is also possible that wild-type PML-RARα may simply promote the cytosolic retention of N-CoR by masking its nuclear localization signal (NLS), which the ∆C-C or AHT mutants cannot do. However, the cytosolic retention of the PML-RARα/N-CoR complex is not due to that the PML-RARα molecule lacks the nuclear localization signal (NLS) of PML, which is located in the C terminal region of PML, because an artificial PML-RARα protein containing the fulllength PML molecule [PML(full-length)-RARα} was also found to be localized in the cytosol along with N-CoR (data not shown). So far, only secreted and membrane proteins appear to be degraded by the ERAD pathway and thus the postulated movement of a nuclear factor to the ER for degradation may appear to be unusual.
However, some transcription factors, including Fos, have also been found in the ER (39). Furthermore, it was recently shown the N-CoR/TAB2 complex is exported to the cytosol in response to IL-1β (40). Thus, our proposal that the N-CoR protein which has the abnormal conformation moves to the ER may not be unusual.
The Ring finger domain serves as a catalytic domain for the ubiquitin ligase activity of many E3 ligases (41). However, in our present study, we found that the in vivo ubiquitination of PML-RARα-induced N-CoR does not require an intact Ring finger of PML-RARα (Fig. 6A). It was recently demonstrated that the Mdm2-mediated nuclear export of the p53 protein requires the presence of an intact Ring finger domain in the Mdm2 protein (42,43). This suggests that the Ring finger may participate not Khan, M. M. et al.

26
only in the recognition of substrates by E3 ligases but may also be involved in other types of protein-protein interactions. Our findings indicate that PML-RARα with an intact Ring finger domain may be involved in transporting the PML-RARα/N-CoR complex across the ER membrane (Fig. 6). Our results also suggest that the Ring finger domain is essential for insoluble protein aggregate formation, possibly because it targets ubiquitinated proteins to ER membrane structures (Fig. 6). Alternatively, the Ring finger may directly induce the insolubility of mis-folded protein by facilitating the formation of tight multi-protein complexes that have the hydrophobic residues on their surfaces (44), whereas a Ring finger mutant may not be able to form such complexes.
The PML-RARα protein did not exhibit any ubiquitination activity when used with recombinant N-CoR and Ubc6 proteins in in vitro ubiquitination assays (data not shown). Moreover, the Ring finger mutant of PML-RARα was equally efficient in inducing the ubiquitination of N-CoR in vivo (Fig. 6A). These findings preclude a role for PML-RARα as an E3 ligase in N-CoR ubiquitination. Multiple E3 ligases, including Hrd1p/Der3p, Parkin, and CHIP have been reported to be involved in ERAD (45)(46)(47).
However, we found that neither Parkin nor CHIP could induce N-CoR ubiquitination in    Relative cell number