NAD(P)H:quinone Oxidoreductase 1 (NQO1) Competes with 20S Proteasome for Binding with C/EBPα Leading to Its Stabilization and Protection against Radiation-induced Myeloproliferative Disease*

Background: NQO1 protects against myeloproliferative diseases. Results: Radiation-induced NQO1 competes with 20S proteasome for binding with myeloid differentiation factor C/EBPα leading to stabilization of C/EBPα and protection against myeloproliferative diseases. Conclusion: NQO1 is an endogenous factor in stabilization of C/EBPα and protection against myeloproliferative diseases during radiation and chemical stress. Significance: NQO1 might serve as therapeutic target for prevention of hematological diseases. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of NQO1 gene in mice leads to increased susceptibility to myeloproliferative disease. In this report, we demonstrate that NQO1 controls the stability of myeloid differentiation factor C/EBPα against 20S proteasomal degradation during radiation exposure stress. Co-immunoprecipitation studies showed that NQO1, C/EBPα, and 20S all interacted with each other. C/EBPα interaction with 20S led to the degradation of C/EBPα. NQO1 in presence of its cofactor NADH protected C/EBPα against 20S degradation. Deletion and site-directed mutagenesis demonstrated that NQO1 and 20S competed for the same binding region 268SGAGAGKAKKSV279 in C/EBPα. Mutagenesis studies also revealed that NQO1Y127/Y129 required for NADH binding is essential for NQO1 stabilization of C/EBPα. Exposure of mice and HL-60 cells to 3 Grays of γ-radiation led to increased NQO1 that stabilized C/EBPα against 20S proteasomal degradation. This mechanism of NQO1 regulation of C/EBPα may provide protection to bone marrow against adverse effects of radiation exposure. The studies have significance for human individuals carrying hetero- or homozygous NQO1P187S mutation and are deficient or lack NQO1 protein.

NAD(P)H:quinone oxidoreductase 1 (NQO1) 2 is a stress-inducible flavoprotein involved in cellular defense against the electrophilic and oxidizing metabolites (1)(2). Induction or depletion of NQO1 levels are associated with decreased and increased susceptibilities to oxidative stress, respectively (3)(4). NQO1 is known to bind to and stabilize tumor suppressor p53, p73␣, and p33 against 20S proteasomal degradation (5-6). These findings suggest that NQO1 exercises a selective "gatekeeping" role in regulating the proteasomal degradation of certain proteins.
Hematopoiesis is a highly orchestrated multi-step process that turns the hematopoietic stem cells into various specialized and distinct blood cell types (7). This process is regulated by transcription factors including C/EBP␣ and PU.1 (7). C/EBP␣ is found predominantly in immature myeloid cells, whereas both lymphoid and myeloid cells express PU.1 (8 -9). C/EBP␣ is a leucine zipper factor (10 -11). Deregulation of C/EBP␣ has been found to be associated with myeloid transformation (12)(13). The PU.1 gene contains two enhancer elements located 14 kilobases upstream of its promoter that bind C/EBP␣ and regulate expression of PU.1 (14).
A cytosine to thymidine (C3 T) polymorphism in exon 6 of human NQO1 gene localized on chromosome 16q22 produces a proline to serine (P187S) substitution that destabilizes the enzyme which is degraded by proteasomes (15)(16). About 2-4% of the normal population is homozygous and 23% is heterozygous for NQO1P187S mutant allele (17). NQO1P187S has been associated with greater risk of neutropenia in benzene-exposed adult Chinese workers (18) and is significantly overexpressed in chemotherapy-related (19) and de novo leukemias (20) in adults. Wiemels et al. (21) reported that NQO1P187S conferred susceptibility to infant acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) with MLL translocations in a British population. Smith et al. (22) reported similar findings in United States. Recently, Eguchi-Ishimae et al. reported association of NQO1P187S with pediatric ALL with MLL-AF4 fusion genes in Japan (23). A more recent study reports NQO1P187S allele as a possible risk and prognostic factor for CLL (24). NQO1 Ϫ/Ϫ mice demonstrated myelogenous hyperplasia (25). Studies revealed that exposure to ␥-radiation resulted in myeloproliferative disease in NQO1 Ϫ/Ϫ mice (26). NQO1 Ϫ/Ϫ mice exposed to ␥-radiation demonstrated bone marrow hypercellularity and enlarged lymph nodes and spleen. The spleen showed disrupted follicular structure, loss of red pulp, and granulocyte and megakarocyte invasion. The above studies demonstrated a role of NQO1 in protection against myeloproliferative diseases including leukemia and raised questions regarding the mechanism of this protection.
In this report, we demonstrate that NQO1 protects myeloid differentiation factor C/EBP␣ against 20S proteasomal degradation. This leads to stabilization of C/EBP␣ and protection against myeloproliferative diseases including leukemia. NQO1 achieves this function by competing with 20S proteasomes for binding with C/EBP␣. Further studies identified the C/EBP␣and NQO1-interacting domains and determined the mechanism of protection.

EXPERIMENTAL PROCEDURES
Materials-Purified 20S, Flag, and mouse NQO1 antibodies, Easyview Red Anti-Flag M2 affinity gel, and MG132 were obtained from Sigma (St. Louis, MO). Human NQO1 and C/EBP␣ antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PU.1 antibody was from cell signaling (Beverly, MA). Anti-20S proteasome core subunits rabbit pAB (Cat. No. ST1053) was purchased from Calbiochem (La Jolla, CA). This antibody recognizes the 20S proteasome core subunits (␣5, ␣7, ␤1, ␤5i, and ␤7) as reported in the company data sheet. Anti-20S proteasome ␣5-subunit mouse mAb (MCP196) (Cat. No. ST1051) was obtained from Calbiochem. It recognizes 20S proteasome ␣5-subunit protein as reported in the company data sheet. V5 antibody was from Invitrogen (Carlsbad, CA). The specificities of antibodies are reported on the company web sites. In addition, the specificities of antibodies were also tested in our laboratory. The antibodies used detected a single band of correct molecular size. Many of the antibodies used in the present report have also been used in our previous publications (25)(26)(27)(28)(29). Nucleofector II and reagents were from Lonza (Cologne, Germany).
Construction of Plasmids-All truncated constructs of mouse C/EBP␣ were engineered by PCR followed by digestion with XbaI and XhoI and subsequently cloning into pCMX vector containing double Flag-tag. Site directed mutations of mouse C/EBP␣ cDNA were generated by first performing two PCRs to amplify the 5Ј-fragment of mouse C/EBP␣ cDNA (carrying EcoRI site) and the rest 3Ј-fragment of mouse C/EBP␣ cDNA (with site mutations and carrying XhoI site), after blunt-end ligation, the fragments with size of about 1080 base pairs were digested with EcoRI and XhoI and subsequently cloned into pCMX vector. This strategy was applied since C/EBP␣ is highly GC rich (72%) and full-length PCR was unsuccessful to amplify the DNA.
␥-Radiation Treatment-Mice and cells were either non-irradiated or irradiated with 1, 3, or 6 Grays of ␥-radiation, waited for 12, 24, and 48 h and analyzed. However, 3 Grays and 48 h that gave the best results are described. Mice were non-irradiated or irradiated with 3 Grays ␥-radiation (Mark I Irradiator, cesium-137, J.L. Shepherad and Associates). Forty-eight hours later, femurs were removed and bone marrow flushed out with cold PBS. The cells were centrifuged and lysed with cold buffer. For ex vivo analysis, mice were sacrificed, and their bone marrow cells were obtained in RPMI/10% FBS. Cells were irradiated with 3 Grays ␥-radiation and cultured in 12-well plates for 48 h for further analysis.
Transfection-Transfection of HL-60 cells with plasmid DNA and U937 or KG-1 cells with siRNA were performed using Amaxa transfection device, Lonza Cell Line Optimization Nucleofector Kit and manufacturer's instructions.
Immunoprecipitation-Cells were lysed with buffer (150 mmol/liter NaCl, 25 mmol/liter Tris, pH 7.4, 1 mmol/liter EDTA, and 1% perfluorooctanoic acid) with protease inhibitor mixture (Roche). 1 mg lysates were precleared with washed protein A/G-agarose beads for 1h at 4°C, followed by bead removal and immunoprecipitation overnight with 20S core subunit antibody (1:100), V5 antibody (1:1000), or antibodies against NQO1, C/EBP␣, Pu.1 and unrelated protein Nrf2. The next day, fresh A/G-agarose beads were added and rotated for 4 h to bind antibodies, then, the samples were washed twice in lysis buffer, boiled for 5 min in 2ϫ SDS loading buffer, and analyzed by SDS-PAGE. Immunoprecipitation of Flag-tagged proteins was done by Easyview Red anti-Flag M2 affinity gel according to manufacturer's protocol.
Ubiquitination Assay-HL-60 cells were co-transfected with 0.5 g of HA-Ub and 1 g of wild type C/EBP␣-V5 or mutant C/EBP␣K276A-V5 or C/EBP␣K277A-V5 or ubiquitination inactive Bcl2K17A-V5. The cells were lysed 24 h later, and 1 mg of cell lysates were immunoprecipitated with anti-V5 antibody. After SDS-PAGE and Western blotting, the PVDF membrane was probed with anti-HA antibody.
In Vitro Translation and 20S Proteasomal Degradation of Translated Protein-1 g V5-tagged NQO1 or NQO1 mutants or C/EBP␣ plasmid DNAs were translated and 2 l from each product were incubated together with purified 20S proteasome in degradation buffer (100 mmol/liter Tris-Cl (pH 7.5), containing 150 mmol/liter NaCl, 5 mmol/liter MgCl 2 , and 2 mmol/ liter DTT) for various time points and quenched via freezing at Ϫ80°C. Samples were separated on 12% SDS-PAGE and immunoblotted. In similar experiments, in vitro translated NQO1 without or with NADH and dicoumarol were included and 20S degradation of C/EBP␣ experiments repeated. In related experiments, in vitro translated C/EBP␣ was incubated with increasing concentration of in vitro translated NQO1 and constant amount of purified 20S proteasome to demonstrate that NQO1 directly competes with 20S for C/EBP␣ binding.
Real-time PCR-RNA was extracted from mice bone marrow cells with or without ␥-radiation and HL-60 cells either transfected with increasing concentration of pcDNA-NQO1-V5 plasmid or NQO1 siRNA using RNeasy mini kit (Qiagen). RNA was converted to cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer's instructions. cDNA was used with Taqman Master Mix and NQO1 (ID: Mm01253561-m1), C/EBP␣ (ID: Mm00514283-S1), PU.1 (ID: Mm00488428-m1), or control GusB (ID: Mm00446953-m1) Primer and Probe. Final mixture was run on 7500 Real Time System (Applied Biosystems) using relative quantitation according to the manufacturer's instructions.
Statistical Analysis-The statistical analyses (student t test and ANOVA) were done by Microsoft Excel and Graphpad Prism. Differences were considered significant at p Ͻ 0.05.

NQO1 Protects C/EBP␣ against 20S Proteasomal
Degradation-Wild type (WT) and NQO1 Ϫ/Ϫ mice were non-irradiated or irradiated with 3 Grays ␥-radiation, bone marrow collected, and immunoblotted (Fig. 1A). Bone marrow cells from non-irradiated NQO1 Ϫ/Ϫ mice showed higher levels of C/EBP␣ (1.5 times) and PU.1 (2 times) as compared with WT mice. This suggested that increased C/EBP␣ and PU.1 were responsible for myeloid hyperplasia in NQO1 Ϫ/Ϫ mice as reported earlier (25). Interestingly, exposure to 3 Grays of ␥-radiation increased NQO1, C/EBP␣ and PU.1 to varying levels in WT mice but failed to do so in NQO1 Ϫ/Ϫ mice bone marrow. These results indicated that NQO1 Ϫ/Ϫ mice were incapable of inducing myeloid differentiation factors C/EBP␣ and PU.1 upon exposure to ␥-radiation, as observed with WT mice.
It is noteworthy that non-irradiated NQO1 Ϫ/Ϫ mice expressed higher C/EBP␣ and Pu.1, as compared with WT mice. Real time PCR analysis showed that this is due to increased transcription of C/EBP␣ in NQO1 Ϫ/Ϫ mice, as compared with wild type mice (supplemental Fig. S1). This might have been to compensate the loss of C/EBP␣ in NQO1 Ϫ/Ϫ mice.
Next, we performed ex vivo bone marrow studies to investigate the role of protein degradation and/or transcription in lack of induction of C/EBP␣ and PU.1 in response to ␥-radiation. Bone marrow cells from WT and NQO1 Ϫ/Ϫ mice were exposed to 3 Grays ␥-radiation in absence and presence of proteasome inhibitor MG132 or epoxomycin and analyzed for p53, C/EBP␣, PU.1, NQO1, and actin. p53 was included as control of MG132-mediated inhibition of proteasomes as reported earlier (Ref. 29, Fig. 1B, left upper panels). MG132 treatment protected C/EBP␣ in NQO1 Ϫ/Ϫ mice from proteasomal degradation (Fig.  1B, left lower panels and the bar graph). This was evident from equal levels of C/EBP␣ in bone marrow cells from WT and NQO1 Ϫ/Ϫ mice exposed to ␥-radiation. Similar results were obtained with another proteasome inhibitor epoxomycin (Fig.  1B, right panels). The results together suggested that NQO1 is required for the stabilization and induction of C/EBP␣ in response to ␥-radiation. PU.1 showed similar alterations as C/EBP␣ because C/EBP␣ is known to regulate PU.1 transcription (14). In related experiments, RNA was analyzed to determine the contribution of transcription in down-regulation of PU.1. The results revealed that exposure to ␥-radiation significantly down-regulated PU.1 gene transcripts in NQO1 Ϫ/Ϫ mice (supplemental Fig. S1). This was expected since C/EBP␣ is known to regulate PU.1 transcription (14). In the same experiment, bone marrow cells from WT mice showed up-regulation of C/EBP␣ and PU.1 in response to radiation exposure. Interestingly, the C/EBP␣ RNA was also down regulated in response to radiation exposure in bone marrow cells from NQO1 Ϫ/Ϫ mice. This was also expected since C/EBP␣ is known to autoregulate its own transcription (30 -31). These results indicated that radiation-mediated degradation of C/EBP␣ in NQO1 Ϫ/Ϫ mice led to decreased transcription of C/EBP␣ that contributed to down-regulation of C/EBP␣ protein. In related experiments, MG132 was used to determine if it has an effect on C/EBP␣ and PU.1 transcription. The results showed that MG132 had no effect on C/EBP␣ and PU.1 transcript (supplemental Fig. S2). In conclusion, the above results together reveal that radiationinduced instability/degradation of C/EBP␣ in the absence of NQO1 is a major mechanism leading to down-regulation of C/EBP␣ in NQO1 Ϫ/Ϫ mice bone marrow.

NQO1 Competition with 20S Proteasome for C/EBP␣ Binding
U937 cells transfected with NQO1 siRNA (Fig. 1E) were analyzed for alterations in NQO1 and its effect on C/EBP␣ and PU.1. The results revealed that overexpression of NQO1 in HL-60 cells led to up-regulation of both C/EBP␣ and PU.1 proteins (Fig. 1D). In the same experiment, the over-expression of V5-His-tagged random peptide control failed to affect levels of C/EBP␣ and PU.1 indicating that NQO1 effect on C/EBP␣ and PU.1 was specific (Fig. 1D). In addition, siRNA-mediated inhibition of NQO1 in U937 cells led to down-regulation of both C/EBP␣ and PU.1 proteins (Fig. 1E). The HL60 cells overexpressing NQO1 in Fig. 1D and U937 cells expressing inhibited NQO1 were also analyzed for NQO1, C/EBP␣, and PU.1 RNA to determine the contribution of transcription (supplemental Fig. S3, left and right panels). The results demonstrated NQO1 plasmid dose-dependent increase in NQO1 RNA, no change in C/EBP␣ RNA (C/EBP␣ increase in Fig. 1D is due to stabilization of C/EBP␣), and dose-dependent increase in PU.1 (because of C/EBP␣ stabilization and transcriptional activation of Pu.1). Similarly, the results with NQO1 siRNA demonstrated dosedependent decrease in NQO1 RNA, no change in C/EBP␣ RNA (C/EBP␣ decrease in Fig. 1E is due to degradation of C/EBP␣), and dose-dependent decrease in PU.1 (because of C/EBP␣ degradation and decreased transcription of Pu.1). Therefore, the results together demonstrate that increase in C/EBP␣ in NQO1-overexpressing HL60 cells in Fig. 1D is due to NQO1 stabilization of C/EBP␣ protein. Similarly, decrease in C/EBP␣ FIGURE 1. NQO1 regulates C/EBP␣ and PU.1. A, lack of induction of C/EBP␣ and PU.1 in the bone marrow of NQO1 Ϫ/Ϫ mice exposed to ␥ -radiation. Mice were irradiated with 3 Grays ␥-radiation, bone marrow cells were collected and analyzed by immunoblotting. The bar graph shows relative protein levels after densitometry analysis, * indicates significant changes of protein levels between control and treatment throughout all the figures. B, MG132 and epoxomycin rescues C/EBP␣ and PU.1 from ␥-radiation induced degradation in NQO1 Ϫ/Ϫ cells. Bone marrow cells were exposed to 3 Grays ␥-radiation, then left alone or treated with 20 M MG132 or 50 nM epoxomycin for 10 h. The cells were lysed and immunoblotted. p53 was analyzed as control for MG132 treatment (left panel). C, HL-60 cells express relatively low level of NQO1 compared with U937 cells. D, overexpression of NQO1 up-regulates C/EBP␣ and PU.1. HL-60 cells were transfected with increasing amount of V5-tagged random control peptide (upper panel) or same amounts of NQO1-V5 (lower panel), transfected cells were lysed and immunoblotted. E, siRNA inhibition of NQO1 leads to down-regulation of C/EBP␣ and PU.1. U937 cells were transfected with control or NQO1 siRNA and cell lysates were immunoblotted. F, NQO1 protects C/EBP␣ against 20S proteasomal degradation. In vitro translated C/EBP␣ and NQO1, purified 20S proteasome and NADH were mixed in combinations as shown for 1 h at 37°C. 5 l mixtures from different treatment were immunoblotted. G, ␥-radiation destructs C/EBP␣ and PU.1 in KG-1 cells with NQO1 knock-down. KG-1 cells were transfected with control or NQO1 siRNA, and 60 g cell lysates were immunoblotted (left panel). KG-1 cells with NQO1 knock-down were irradiated with 3 Grays ␥-radiation, then analyzed by immunoblotting (middle panel and bar graph). Molecular weight markers are shown on the right side of panels in A. DECEMBER 7, 2012 • VOLUME 287 • NUMBER 50 in U937 cells expressing siRNA-mediated inhibition of NQO1 is due to degradation of C/EBP␣ protein.

NQO1 Competition with 20S Proteasome for C/EBP␣ Binding
NQO1 is known to stabilize p53 against 20S proteasomemediated ubiquitination-independent degradation (5,29). Therefore, we performed in vitro assays to investigate the role of 20S proteasome in C/EBP␣ degradation and NQO1 protection against 20S degradation of C/EBP␣. Incubation of in vitro translated C/EBP␣ with purified 20S showed significant degradation of C/EBP␣ within 1 h (Fig. 1F). Inclusion of in vitro translated NQO1 partially protected C/EBP␣. Interestingly, NQO1 in combination with its cofactor NADH in the same experiment provided complete protection of C/EBP␣ against 20S proteasomal degradation (Fig. 1F). The results collectively indicated that NQO1 stabilizes C/EBP␣ against 20S proteasomal degradation. We also used in vitro translated NQO1-V5 and Flag-C/EBP␣ proteins and purified 20S proteasome to investigate if NQO1 competed with 20S proteasome for C/EBP␣ leading to stabilization of C/EBP␣ against 20S degradation. The results are shown in supplemental Fig. S4. The results reveal that NQO1 directly competed with 20S proteasome for C/EBP␣ protein. Increase in NQO1 led to NQO1 concentration-dependent increase in NQO1:C/EBP␣ interaction and decrease in 20S:C/EBP␣ interaction (supplemental Fig. S4).
The studies were extended to CD34 positive human myeloid KG-1 cells that upon transfection with NQO1 siRNA demonstrated siRNA concentration dependent decrease in NQO1, C/EBP␣ and PU.1 (Fig. 1G, left panels). In related experiments, NQO1 siRNA-mediated inhibition of NQO1 in KG-1 cells upon exposure to 3 Grays of ␥-radiation also led to significant decrease in C/EBP␣ and Pu.1 (Fig. 1G, middle panels). These results revealed that NQO1 also controls C/EBP␣ and downstream Pu.1 in human myeloid KG-1 cells.
WT and NQO1 Ϫ/Ϫ mice non-irradiated or irradiated with 3 Grays of ␥-radiation were also analyzed 48 h after exposure for Gr-1-positive mature granulocytes and apoptosis in granulocytes in bone marrow (supplemental Fig. S5) and blood CBCs (supplemental Fig. S6). Bone marrow analysis revealed radiation-mediated 7-fold decrease in granulocytes in NQO1 Ϫ/Ϫ mice as compared with only a 2.2-fold decrease in WT mice (supplemental Fig. S5). In the same experiment, apoptosis in granulocytes was increased by only 1.6-fold in NQO1 Ϫ/Ϫ compared with 2-fold in WT mice (supplemental Fig. S5). These results clearly suggested that differentiation of granulocytes in NQO1 Ϫ/Ϫ mice was significantly decreased as compared with WT mice upon exposure to ␥-radiation. Blood CBCs analysis also showed only 3.5-fold increase in neutrophils in NQO1 Ϫ/Ϫ mice exposed to radiation as compared with 6.5-fold increase in WT mice, thus supporting the above observations (supplemental Fig. S6). Collectively, these results demonstrated that absence of NQO1 upon exposure to ␥-radiation led to loss of induction of C/EBP␣ and Pu.1 and decreased granulocyte differentiation.
NQO1 and 20S Both Interact with C/EBP␣ but Not with PU.1-Immunoprecipitation assays were performed to investigate the role of NQO1 and 20S interaction with C/EBP␣ in inhibition of 20S degradation of C/EBP␣. Bone marrow from WT and NQO1 Ϫ/Ϫ mice, HL-60 cells and HL-60 cells transfected with NQO1-V5, Flag-C/EBP␣ and an unrelated Nrf2 plasmids were analyzed for interactions among C/EBP␣, 20S, and NQO1 ( Fig.  2A). The transfected HL60 cells were also immunoprecipitated with anti-Nrf2 antibody as negative control of immunoprecipitation experiments. The results showed that all three (C/EBP␣, 20S, and NQO1) proteins interacted with each other in WT mice (left panels), HL-60 (middle panels), and transfected HL-60 cells (right panels). The results further showed that C/EBP␣ interacted with 20S in absence of NQO1 in NQO1 Ϫ/Ϫ mice bone marrow. It is noteworthy that unrelated Nrf2 protein as expected failed to interact with NQO1, C/EBP␣ or 20S ( Fig.  2A, right panels). This indicated that interactions among C/EBP␣, NQO1, and 20S were specific. To further define the interactions, we used in vitro translated Flag-C/EBP␣ and NQO1-V5 and purified 20S proteins and performed immunoprecipitation assays to investigate the mutual interaction between two of the three proteins in the absence of the third one. Results indicated that any two of the three proteins interacted with each other independent of the third one (Fig. 2B). These results collectively demonstrated that NQO1 and 20S both interact with C/EBP␣ besides NQO1 interaction with 20S. The results also showed that interactions of two of the three proteins are independent of the third one. In similar experiments as described above for C/EBP␣, immunoprecipitation assays failed to show PU.1 interaction with either NQO1 or 20S proteasomes (Fig. 2C). This confirmed that NQO1 controls PU.1 through C/EBP␣ that regulates transcription of PU.1.

NQO1 Competition with 20S Proteasome for C/EBP␣ Binding
tein containing internal deletion of protein domain between amino acids S268-V279 failed to interact with both NQO1 and 20S (Fig. 3D, right panels). These results together revealed that both NQO1 and 20S proteasome interacted with the same domain in C/EBP␣ protein between amino acid S268-V279. To confirm this, three C/EBP␣ fragment peptides including S268-V279 (full-length), S268-G273 (N-terminal half), and K274-V279 (C-terminal half) were synthesized and used in in vitro assays to determine their effect on C/EBP␣ interaction with 20S (Fig. 3E). All three peptides competed with C/EBP␣ for binding with 20S and degradation. Interestingly, C-terminal half peptide containing basic lysine residues were significantly more efficient in competing C/EBP␣ against 20S degradation, as compared with N-terminal half containing small/neutral amino acids.
Lysine 276 and Lysine 277 of C/EBP␣ Play Critical Roles in Its Interaction with NQO1-Alignment of mouse, human, and rat C/EBP␣ domains between amino acid 268 -279 showed conserved residues K274, K276, and K277; G269, G271, and 273; A272 and A275; S278; and V279 (Fig. 4A). Among these, basic lysine residues appeared most likely to interact with NQO1 and 20S. Site-directed mutagenesis was used to individually mutate K274, K276, and K277 to generate Flag-tagged C/EBP␣ K274A, K276A, and K277A mutant plasmids. Transfection and immunoprecipitation assays demonstrated that all C/EBP␣ mutants, NQO1-V5 and 20S were successfully immunoprecipitated (Fig.  4B, left, middle, and right panels). Transfection and forward/ reverse immunoprecipitation experiments also showed that mutation of K276 and K277 to alanine resulted in the loss of interaction of C/EBP␣ with NQO1 (Fig. 4B, left and middle panels). On contrary, C/EBP␣ K274A mutant still interacted with NQO1 (Fig. 4B, left and middle panels). Based on these results, we generated double mutant C/EBP␣ K276A/K277A that also failed to interact with NQO1 (Fig. 4B, left and middle  panels). These results suggested that K276/K277 of C/EBP␣ is required for its interaction with NQO1. Interestingly, all four C/EBP␣ mutants K274A, K276A, K277A, and double mutant K276A/K277A interacted with 20S in a similar capacity as normal C/EBP␣ (Fig. 4B, right panels). We also generated G273A and S278A that showed no effect on C/EBP␣ interaction with NQO1 or 20S (Fig. 4B). These results indicated that mutations in individual amino acid between S268 and V279 had no effect on C/EBP␣ interaction with 20S, suggesting the requirement of all of the amino acids in 268 -279 domain for interaction of C/EBP␣ with 20S.

NQO1 C-and N-terminal Deletions Result in Loss of Interaction with C/EBP␣-NQO1 protein contains 8 ␣-helixes and 9
␤-sheets (32-33). V5-tagged C-and N-terminal deletions were generated to identify the protein domain(s) required for interaction with C/EBP␣ and 20S. Transfection of these mutants in HL-60 cells produced expected size of truncated NQO1-V5 proteins (Fig. 5A). Immunoprecipitation assays revealed that all the deletions of NQO1 failed to interact with C/EBP␣ or 20S proteasome (Fig. 5B). This suggested that full-length NQO1 protein is required for interaction with C/EBP␣ and 20S.
Disruption of NADH Binding to NQO1 Abolished the Interaction between NQO1 and C/EBP␣-Dicoumarol and NQO1Y127A/Y129A double mutant were used in in vivo and in vitro studies to disrupt NQO1 binding with NADH to determine its effect on NQO1 interaction with C/EBP␣ and 20S (in vivo studies) and protection of C/EBP␣ against 20S degradation (in vitro studies). Dicoumarol is a competitive inhibitor of NADH binding to NQO1 and NQO1Y127/Y129 is required for NADH binding (32)(33). The results showed that both dicoumarol and double mutant NQO1Y127A/Y129A disruption of NADH binding with NQO1 led to the loss of NQO1 binding with C/EBP␣ but not with 20S (Fig. 6, A and B). In vitro translation/degradation assays showed that dicoumarol and double mutant NQO1Y127A/Y129A both inhibited NQO1 from protection of C/EBP␣ against 20S degradation (Fig. 6C). These results revealed that NADH binding to NQO1 is essential for NQO1 protection of C/EBP␣ against 20S degradation. In addition, the NADH binding to NQO1 is not required for NQO1 interaction with 20S.
NQO1P187S Mutation Leads to Degradation of NQO1 and Upon Stabilization Protects C/EBP␣ against 20S Degradation-NQO1P187S mutant is an unstable protein and is degraded by proteasomes (15)(16). HL-60 cells transfected with mutant plasmid expressed low level of NQO1P187S mutant protein that was significantly stabilized in presence of proteasome inhibitor MG132 (Fig. 6D, left panels). Interestingly, addition of V5-tag at C terminus of mutant NQO1P187S protein stabilized the protein even in the absence of MG132 presumably due to conformational changes (Fig. 6D, right panels). In

. C/EBP␣ domain between S268 and V279 is required for its interaction with both NQO1 and 20S in HL-60 cells.
A, schematic domain structure of C/EBP␣ and its truncated mutants. DBD, DNA binding domain; LZD: leucine zipper region, and 2ϫFlag-tag are indicated. B, input for co-immunoprecipitation. Flag-tagged wild type and mutant C/EBP␣ plasmids were transfected into HL-60 cells, 80 g cell lysates were loaded and separated on 12.5% SDS-PAGE, and probed with anti-Flag antibody. C, C/EBP␣ S268-V279 domain is required for interaction with both NQO1 and 20S. HL-60 cells were co-transfected with full-length or truncated mutant Flag-C/EBP␣ and NQO1-V5, cell lysates were immunoprecipitated with antibodies as indicated, immunoblotted, and probed with anti-Flag, anti-V5, and anti-20S antibodies as shown. D, internal deletion of S268-V279 domain from C/EBP␣ leads to loss on interaction of C/EBP␣ with NQO1 and 20S. HL-60 cells were transfected with full-length or internal deletion mutant of Flag-C/EBP␣ and NQO1-V5, lysed, immunoprecipitated, and immunoblotted. Immunoblot was probed with anti-Flag and anti-V5 antibodies (right upper panels), or probed with anti-Flag and anti-20S antibodies (right lower panels). E, in vitro translated wild type C/EBP␣ is incubated with purified 20S proteasomes in absence and presence of the synthesized peptides C/EBP␣ (S268-V279, S268-G273, and K274-V279) for 2 h and analyzed by immunoblotting and probing with anti-C/EBP␣ antibody. Molecular weight markers are shown on the right side of panels. All input were 10% of the proteins used in the experiments.

NQO1 Competition with 20S Proteasome for C/EBP␣ Binding
vitro translation and degradation assays revealed that NQO1P187S-V5 protein protected C/EBP␣ against 20S degradation (Fig. 6E). These results indicated that it is instability/loss not P187S mutation of NQO1 protein that associates with adverse effects.
␥-Radiation Increases NQO1 Interaction with C/EBP␣ and Decreases Interaction between 20S and C/EBP␣-HL-60 cells were non-irradiated or irradiated with ␥-radiation and analyzed for C/EBP␣, NQO1, and 20S interactions by immunoprecipitation and immunoblotting (Fig. 7A, left panels). The exper-   ). B, V5-tagged NQO1 deletion mutants were co-transfected with Flag-C/EBP␣. The transfected cells were lysed and immunoprecipitated with IgG (control), anti-Flag and anti-V5. Immunoprecipitated proteins were separated on SDS-PAGE and immunoblotted. The immunoblots were probed with anti-V5 to detect NQO1 deletions, anti-Flag to detect Flag-C/EBP␣ and anti-20S 5␣ to detect 20S. All input were 10% of the proteins used in the experiments. DECEMBER 7, 2012 • VOLUME 287 • NUMBER 50 iment was repeated three times and densitometry analysis of bands performed. The data are plotted in a bar diagram (Fig. 7A,  right panel). The results demonstrated significant increases (108%) in NQO1 interaction with C/EBP␣ in response to ␥-radiation (Fig. 7A). The results also revealed ␥-radiation-induced significant decrease (-42%) in 20S interaction with C/EBP␣ (Fig. 7A). In the same experiments, the exposure to ␥-radiation only marginally increased NQO1 interaction with 20S (Fig. 7A).

NQO1 Competition with 20S Proteasome for C/EBP␣ Binding
In similar experiments, WT mice were exposed to 3 Grays of ␥-radiation, bone marrow collected and analyzed for C/EBP␣, NQO1, and 20S interaction (Fig. 7B). The results from mice studies supported HL-60 cell data. Similar increases (110%) in NQO1 interaction with C/EBP␣ and decreases (Ϫ56%) in 20S interaction with C/EBP␣ were observed. The results together suggested that exposure to ␥-radiation led to significantly greater interaction of C/EBP␣ with NQO1 and reduced interaction with 20S proteasomes leading to stabilization of C/EBP␣ and up-regulation of downstream gene expression. The NQO1 interaction with 20S was marginally increased in HL-60 cells and mice exposed to ␥-radiation.

DISCUSSION
NQO1 protects against myeloproliferative diseases including leukemia (19 -26). We demonstrate here that NQO1 controls myeloid differentiation factor C/EBP␣ and PU.1 to provide this protection. Disruption of C/EBP␣ and PU.1 both are known to be associated with development of myeloid leukemia (11)(12)34). WT mice upon exposure to ␥-radiation showed significant induction of C/EBP␣ and PU.1 and myeloid differentiation (current report) and absence of myeloproliferative disease (26). On the other hand, NQO1 Ϫ/Ϫ mice exposed to ␥-radiation showed lack of induction of both C/EBP␣ and PU.1 and myeloid differentiation (current report) and development of myeloproliferative disease (26). Therefore, it is reasonable to suggest that the lack of induction of C/EBP␣ and PU.1 contributed to significantly decreased differentiation of myeloid cells and myeloproliferative diseases in NQO1 Ϫ/Ϫ mice. Lack of induction of C/EBP␣ was due to rapid degradation by 20S proteasomes in the absence of NQO1. NQO1 directly interacted with C/EBP␣ and prevented its degradation by 20S proteasomes. This led to stabilization of C/EBP␣, normal differentiation of myeloid progenitor cells and protection against myeloproliferative diseases. NQO1 indirectly regulates PU.1 since PU.1 does not directly interact with NQO1 or 20S proteasomes. C/EBP␣ is known to up-regulate PU.1 gene transcription (14). Therefore, NQO1 mediated stabilization or 20S degradation of C/EBP␣ up-or down-regulated PU.1 transcription, respectively.
NQO1 and 20S proteasomes interacted and competed for the same C/EBP␣ domain 268 SGAGAGKAKKSV 279 . Internal deletion of this domain from C/EBP␣ resulted in the loss of interaction with both NQO1 and 20S. The NQO1 interaction with C/EBP␣ was further narrowed down to two lysine residues at amino acid position 276 and 277. Mutation of one or both of these lysine residues led to the complete loss of C/EBP␣ interaction with NQO1 but not with 20S. Interestingly, single amino acid mutations in the 20S interacting domain had no effect on CEBP␣ interaction with 20S. This indicated that complete domain was required for C/EBP␣ and 20S interaction. The NQO1 and 20S interacting domain in mouse C/EBP␣ characterized by us was found highly conserved in human and rat. Deletions in NQO1 protein had deleterious effect on NQO1 interaction with C/EBP␣ and 20S. This indicated that secondary/tertiary/folding structures are required for NQO1 interaction with C/EBP␣ and 20S. NADH was required for NQO1 binding with C/EBP␣ but not for that with 20S. This was clearly evident from the following. Dicoumarol that competes with NADH for binding with NQO1 abolished NQO1 interaction with C/EBP␣. In addition, mutant NQO1Y127A/Y129A deficient in binding with NADH failed to interact with C/EBP␣. Therefore, NADH binding with NQO1 regulates NQO1 control of stabilization of C/EBP␣. It is possible that radiation/ chemical stress-induced intracellular redox changes leads to higher levels of NADH which binds with NQO1 that in turn interacts with C/EBP␣ leading to stabilization of C/EBP␣. NQO1 belongs to a battery of cytoprotective genes that coordinately activate in response to radiation and chemical stress (Ref. 29, present studies). This induction is critical in protection FIGURE 6. Disruption of NADH binding to NQO1 abolished the interaction between NQO1 and C/EBP␣. A, dicumarol disrupts C/EBP␣ binding with NQO1. HL-60 cells were co-transfected with Flag-C/EBP␣ and NQO1-V5 and treated with 20 M dicumarol. Cell lysates were immunoprecipitated and probed with anti-V5, anti-Flag, or anti-20S antibodies. B, mutation of NQO1Y127/Y129 abolished its interaction with C/EBP␣. HL-60 cells were cotransfected with Flag-C/EBP␣ and NQO1(Y127/129A)-V5, cell lysates were immunoprecipitated and immunotblotted. C, NADH binding to NQO1 is essential for NQO1 stabilization of C/EBP␣ against 20S degradation. In vitro translated C/EBP␣ was incubated with purified 20S proteasomes in absence and presence of NQO1-V5, NQO1-Y127A/Y129A-V5, NADH, or Dicoumarol for indicated time periods. The incubation mixtures were analyzed by Western blot. D, NQO1P187S mutant is stabilized by V5-tag. HL-60 cells were transfected with hNQO1P187S-V5 or hNQO1P187S plasmids, 24 h later, 10 M MG132 was included in the media for 10 h. 20 g cell lysates were loaded for SDS-PAGE, and probed with antibodies as indicated. E, NQO1P187S-V5 stabilizes C/EBP␣. In vitro-translated C/EBP␣ was incubated with NQO1P187S-V5, NADH, and 20S proteasome for different time, incubation mixtures were analyzed by Western blot. All input were 10% of the proteins used in the experiments.

NQO1 Competition with 20S Proteasome for C/EBP␣ Binding
against myeloproliferative disease exposed to ␥-radiation (26). The induction of NQO1 enhances its interaction with C/EBP␣ and reduces 20S interaction with C/EBP␣ leading to protection and stabilization of C/EBP␣. NQO1P187S mutation is known to destabilize NQO1 because of its degradation by proteasomes (15)(16). It was interesting to observe that addition of V5-tag at the C-terminal end of the protein led to stabilization of NQO1Y187S-V5 mutant protein. This mutant interacted with C/EBP␣ and 20S in similar capacity as normal NQO1. This led to conclusion that higher susceptibility of human individuals carrying NQO1P187S mutation to develop myeloproliferative diseases including leukemia is due to loss of NQO1 protein and not due to lack of interaction with C/EBP␣ and 20S. Studies have shown that homozygous and heterozygous population for NQO1P187S mutation (17,21,23) might be at risk for development of myeloproliferative diseases.
A model of NQO1 protection of C/EBP␣ against 20S degradation is shown (supplemental Fig. S8). Under normal conditions, both 20S and NQO1 interact with C/EBP␣ and control physiological level of C/EBP␣, PU.1 and myeloid differentiation. Exposure to ␥-radiation stress led to increased NQO1 and increased NQO1 interaction with C/EBP␣. Exposure to ␥-radiation also reduced 20S:C/EBP␣ interaction. Increased NQO1 interaction with C/EBP␣ and decreased interaction of C/EBP␣ with 20S led to stabilization of C/EBP␣, increased expression of PU.1 and differentiation of myeloid cells and protection against ␥-radiation. On contrary, loss of NQO1 leads to increased 20S interaction with C/EBP␣, degradation of C/EBP␣, down-regulation of PU.1, lack of myeloid cell differentiation, and myeloproliferative diseases including leukemia.