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Manuscript M2:02286 (revised) April 10, 2002

Mutational analysis of human thioredoxin reductase 1: Effects on p53 mediated gene expression and Interferon and

Retinoic acid induced cell death

Xinrong Ma, Junbo Hu1, Daniel J. Lindner2, and Dhananjaya V. Kalvakolanu*

Greenebaum Cancer Center, Department of Microbiology & Immunology, Molecular and Cellular Biology Program, University of Maryland School of Medicine, Baltimore, MD 21201. Running title: thioredoxin reductase in tumor suppression

* Corresponding author

Phone: 410-328-1396 Fax: 410-328-1397 E-mail: dkalvako@umaryland.edu 1 Contributed Equally to this work

Current address: 1 Department of Surgery, Tongji Medical Center, Wuhan, P.R. China 2Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, OH 44195

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 12, 2002 as Manuscript M202286200 by guest on N

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Abstract:

The IFN-β and all-trans retinoic acid (RA) combination suppresses tumor growth by inducing

apoptosis in several tumor cell lines. A genetic technique permitted the isolation of human

thioredoxin reductase (TR) as a critical regulator of IFN/RA induced cell death. Our recent

studies have shown that TR1:Trx1 regulated cell death is effected in part through the activation

of p53 dependent responses. To understand its death regulatory function, we have performed a

mutational analysis of TR. Human TR1 has 3 major structural domains, the FAD binding (FAD),

NADPH binding (NBD) and an Interface domain (ID) Here, we show that the deletion of the C-

terminal interface domain results in a constitutive activation of TR dependent death responses

and promotes p53 dependent gene expression. TR mutant without the ID still retains its

dependence on Trx for promoting these responses. Thus, our data suggest that TR-ID acts as a

regulatory domain.

Abbreviations used:

GRIM: Gene associated with Retinoid-IFN induced mortality; IFN: Interferon; RA: all trans

retinoic acid; SRB: Sulforhodamine B; STAT: Signal transducing activator of transcription; TR:

thioredoxin reductase; Trx: thioredoxin.

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Introduction:

Interferons (IFN) exert antitumor effects by inducing the expression of a number of

cellular genes using the Janus tyrosine kinase (JAK)-Signal Transducing Activator of

Transcription (STAT) pathways (1,2). A higher susceptibility of IFN-γ receptor-/- and STAT1 -/-

mice to chemical carcinogenesis than their wildtype counterparts, and a failure of syngeneic mice

to reject the IFN-γ receptor-/- and STAT1 -/- tumors underscore the importance of IFNs in tumor

growth control (3). Similarly, two IFN regulated transcription factors, IRF-1 and IRF-8 (ICSBP),

act as tumor growth suppressors (4,5) since mutations in these genes cause leukemias (6,7). In

rodent cells IFN-stimulated transcription factors of the p200 family control cell cycle progression

(8,9). IFNs also downregulate c -myc expression, activate tumor suppressor pRb and inhibit E2F

to inhibit cell cycle progression in human cell lines (10-12). Although a great deal is known

about IFN signaling pathways and the transcription factors involved, very little is known about

the gene products that mediate the tumor suppressive pathways employed by IFNs. Additionally,

despite their beneficial therapeutic effects in certain leukemias, IFNs are marginally active in the

therapy of solid tumors (13,14). Clinical and experimental models have shown that combination

of IFNs with retinoids, a class of vitamin A derivatives, yields a highly effective growth

suppressive effect in several solid tumors (15-17). All trans Retinoic Acid (RA), a vitamin

metabolite, inhibits the growth of promyelocytic leukemias, and teratocarcinomas in vitro (18).

Two structurally similar but genetically distinct classes of transcription factors, the retinoic acid

receptor (RAR) and the retinoid X receptor (RXR) mediate retinoid induced growth suppression

(19). One such receptor RARβ appears to be a tumor suppressor (20,21). However, the identity

of retinoid-regulated growth inhibitory gene products is unknown.

Our earlier studies showed that the IFN−β/RA combination, but not single agents, causes

cell death in vitro and suppresses tumor growth in vivo (17). Using a genetic technique we have

identified several Genes associated with Retinoid-IFN induced Mortality (GRIM) recently (22).

Human thioredoxin reductase 1 (TR1), a redox regulatory enzyme (23,24), was identified as one

of the GRIMs (22). Subsequent studies have shown that TR and its substrate Trx activate cell

death by modulating the activity of caspase-8 and tumor suppressor p53 (25-27). To further

understand the structure-function relationship of TR to these processes, we have performed a

mutational analysis. A comparative analysis of primary structure of this enzyme with other redox

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enzymes led to the assignment of 3 major modules, the FAD, NBD and ID (23,24). While the

FAD and NBD are critical for redox function and well conserved among the TRs from all

sources, the ID is unique to mammalian TR. ID has been suggested to act as a dimerization

surface to generate functional TR (23,24). However, the functional significance of ID has not

been fully appreciated. Here we show that removal of ID enhances death stimulatory activity of

TR and p53 dependent gene expression.

Materials and Methods:

Reagents: Restriction and DNA modifying enzymes (NE Biolabs); G418 Sulfate, IPTG and

Lipofectamine plus (Life Technologies); nylon membranes, ECL reagents and horseradish

peroxidase coupled to anti-rabbit or anti mouse antibodies (Amersham Pharmacia Inc); human

IFN−βser (Berlex Inc.); and mouse monoclonal antibodies against actin, flag epitope (Sigma Inc),

p53 (Oncogene Science Inc) and myc-epitope (Zymed Inc) were employed in these studies.

Rabbit polyclonal antibody against the C-terminal peptide ofTR1 was described earlier (22).

Fresh stocks of all-trans retinoic acid (Sigma) were prepared in ethanol and added to cultures

under subdued light.

Cell culture: MCF-7 cells were cultured in phenol red free EMEM supplemented with 5%

charcoal stripped fetal bovine serum (CSFBS) and 10-11M estradiol during treatment with IFN-β

and all trans retinoic acid (RA). MCF-7 cells stably transfected with wildtype and mutant forms

of Trx were described earlier (25). The mutant Trx bears serine residues in the place of cysteines

at positions 32 and 35 (28). MCF-7 cells stably transfected with mammalian expression vector

pCMV-neo (MCF-7 neo) or the same vector with the E6 gene of human papilloma virus type-16

(MCF-7 E6) were provided by A.J. Fornace Jr., National Cancer Institute, Bethesda, MD (29).

The loss of p53 function in MCF-7 E6 cells has been demonstrated in earlier (29,30). These cells

were grown in phenol red free media 24h before treatments were initiated. DLD human

carcinoma cells, which lack the endogenous p53, were a gift from Bert Vogelstein, Johns

Hopkins University Oncology Center, Baltimore, MD.

Plasmids: Mammalian expression vector, pCMV-flag bears a flag epitope sequence in its

multiple cloning region. An in frame insertion of any cDNA lacking the N-terminal methionine

into this vector generates the protein with a flag epitope tag at the N-terminus. Mammalian

expression vector pCXN2-myc vector carries a C-terminal myc epitope tag. Cloning of an insert

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without a “stop” codon between the 5’ EcoRI and 3’ KpnI sites of this vector permits the

addition of a myc-tag to the expressed protein. p53-luc carries p53 binding sites (8X) cloned

upstream of SV40 early promoter in the pGL3 basic vector (Promega Inc) was described earlier.

Wildtype and mutant p53 (R175 H) cloned in the pCMV expression vector were described

elsewhere (31,32). A luciferase reporter driven by human Bax promoter, Bax-Luc, was provided

by Carol Prives, Columbia University, New York, NY (33).

Generation of TR mutants: Gene specific primers bearing specific restriction enzyme cutting

sites (for facilitating the sub-cloning) and AmpliTaq gold enzyme (Roche-Perkin Elmer) were

employed in PCR for generating TR mutants. All primers used in this study are listed in table 1.

Two separate sets of primers were used for generating myc- and flag- epitope tagged constructs.

Construction of a myc-tagged full length TR was described in our earlier publication (26). At

first the myc-tagged mutants were generated, which served as templates for generating flag-

tagged mutants. Twelve cycles of PCR was performed to avoid the emergence of unwanted

mutants, owing to polymerase errors. Mutants were sequenced to verify their identity.

Mutant ∆-FAD was amplified using myc-primers -2 and -3 with wildtype TR cDNA as

template. ∆-ID was generated using myc primers -1 and -4. For generating the ∆-NBD mutant, 4

primers were used. The myc-primer 1 and ∆-NBD primer 1were used for amplifying the FAD

domain. The myc-primer2 and ∆-NBD primer2 were used for amplifying the ID region.

Oligonucleotides used for the amplification of FAD and ID regions have a BamHI site at their 3’

and 5’ ends, respectively. The myc primers -1 and -2 bore EcoRI and Kpn I sites. The FAD

product was digested with EcoRI and BamHI; and the ID product was digested with BamHI and

KpnI and then purified. The products were combined with pCXN2-myc vector pre-digested with

EcoRI and KpnI in a three-way ligation reaction. The final ∆-NBD construct has two non-

template derived aminoacids, a glycine and a serine, at the junction of FAD and ID, due to a

BamHI site present in amplifying primers. Constructs f -TR and f -∆-NBD were generated using

flag-primers -1 and -2, with the corresponding myc-tagged constructs as templates. The f-∆-ID

and f -∆-FAD mutants were generated using flag primer pairs –1 and –4 and flag primer pairs –3

and –2, respectively. f-tagged truncated mutants of ∆-ID were generated using the indicated

reverse primers and flag primer-1, with wildtype TR as template. Point mutants were generated

using ∆-ID80 as a template. For example, for generating the T193A mutant a reverse and a

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forward primer bearing the same mutation were used in a 3 step PCR. Flag primer 1 and reverse

primer (mutagenic) were used in first PCR reaction. In the other forward primer (mutagenic) and

∆-ID-80 R primer were used for the second PCR. Purified PCR products from 1 st and 2 nd PCR

were mixed, denatured and annealed. This mixture now served as template for flag primer 1 and

∆-ID-80 R primer to generate the final product. The final PCR product was digested with EcoRI

and KpnI and ligated to pCMV-flag. The other mutants were generated in a very similar manner,

using appropriate mutant primers.

Cell growth assay: Cells (2000/well) were seeded into 96-well plates. Drugs were added and

growth was monitored using a colorimetric assay (34). Each group of treatments had 8 replicates.

Cells were fixed with 10% trichloroacetic acid at the end of the experiment and stained with

0.4% Sulforhodamine B (Sigma). The bound dye was eluted with 100 µl of Tris-HCl (pH 10.5)

and absorbance was monitored at 570 nm. One plate was fixed with TCA, 10h after plating.

Absorbance obtained with this plate was considered as 0% growth. Absorbance obtained with

untreated cells was considered as 100% growth. An i ncrease and decrease of A570 values in the

experimental wells relative to the 0% value indicates cell growth and death, respectively.

Death assays: Cell death was determined using Annexin-V binding assays. Following treatment

with IFN/RA, cells were stained using a commercially available kit (Trevigen Inc) per

manufacturer’s recommendation. FITC positive cells were considered as apoptotic and were

quantified using flow cytometry.

Gene expression analyses: Transfection, β-galactosidase, luciferase assays, SDS-PAGE,

Electrophoretic mobility shift analyses (EMSA) were performed as described in our earlier

publications (25-27). Total amount of transfected DNA (1.0 µg) was kept constant by adding

corresponding empty expression vector DNA, where required. In general, 0.2 µg of luciferase

and 0.2µg of TR mutant were cotransfected. CMV β-galactosidase reporter (0.1 µg) was used as

an internal control for normalizing variations in transfection efficiency. Electrophoretic mobility

shift assay with p53 oligonucleotides was performed as described earlier (25-27).

Western blot analysis: Equal quantities of cell extracts were separated on 12% SDS-PAGE and

western blotted onto nylon membranes. Specific first antibodies were incubated with the blots as

described in our previous publications (22). These blots were washed and incubated with an

appropriate second antibody tagged with horseradish peroxidase. Protein bands were visualized

using a commercially available enhanced chemiluminescence (ECL) kit (Amersham Inc).

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Results:

Generation of TR mutants: We have shown earlier that over expression of a catalytically

inactive TR1or a redox inactive Trx1 in human tumor cell lines imparts resistance to IFN/RA

induced cell death (25,26). In contrast, a wildtype TR1 and Trx1 promoted cell death under the

same conditions. However, the role of other TR domains in cell growth control is unknown. To

understand the relationship between structural domains of TR and cell death regulation, we have

generated new TR mutants. Using PCR we generated 3 mutants, ∆-FAD, ∆-NBD and ∆-ID, each

lacking the FAD binding, NADPH binding and the interface domains, respectively. Since no

domain specific antibodies are available for TR1, we have cloned the PCR products into

mammalian expression vectors, pCMV-flag or pCXN2-myc. Proteins expressed from pCMV-

flag and pCXN2-myc will bear a N-terminal flag- and a C-terminal myc- epitope tag,

respectively. Wildtype TR produces a polypeptide with a theoretical Mr of 54.7 kDa. However, it

migrates as a ~58 kDa protein on SDS-PAGE due to post-translational modifications. The ∆-

FAD, ∆-NBD and ∆-ID constructs are expected to yield 40.1, 33.2 and 27.6 and kDa peptides,

respectively. The mutants were transiently transfected into human breast carcinoma cell line

MCF-7 to check for the production of proteins of proper size. Cell lysates were prepared and

western blotted using either Flag- or myc- epitope specific monoclonal antibodies. Indeed, all

mutants can be expressed to a comparable level upon transfection (Figure 1B and C). Both tags

were used only to demonstrate that either N-terminal or C-terminal tags have no effect on protein

function. Furthermore, the pCXN2-myc has a G418 resistance marker for selecting the stably

transfected cells, which is absent from pCMV-flag. The flag tag is shorter than the myc-tag by

about 8 aminoacids.

Removal of ID enhances the cell death effects of TR: To test the effect of TR

mutants on cell growth, we first generated cell lines that stably express them. Since the pCXN2-

myc vector also carries a G418r marker it permits the selection of stable transfectants, we have

used the myc-tagged constructs for this purpose. As observed earlier, transfection of a wildtype

TR resulted in the formation of fewer colonies than the vector. The ∆-FAD construct gave rise to

marginally more colonies than the control vector transfected cultures indicating its inhibitory

effect on cell death. The ∆-NBD mutant produced comparable number of colonies to that of

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vector. Interestingly, the ∆-ID mutant yielded 75% and 50% fewer G418r colonies than the

vector and TR transfected cultures, respectively (Fig. 2A). The G418r colonies in each group

were pooled and used in the experiments described below to avoid a clonal bias. In the next

experiment the effect of TR mutants on cell growth was determined using a colorimetric assay

(34), where cell growth is quantified on the basis of the amount of sulforhodamine B dye bound

to cells (Fig. 2B). This method correlates well with Coulter counting and determination of cell

number. While the ∆-FAD expressing cells grew slightly faster than the vector transfected

cultures, the TR and ∆-ID transfectants grew relatively slower. Expression of ∆-ID caused

significantly slower growth compared to TR. Growth of ∆-NBD transfectants was comparable to

that of vector-transfectants. These observations suggest that removal of ID converts TR into a

significantly more potent inhibitor of cell growth. Similar results were obtained with f -tagged

TR mutants (data not shown).

To demonstrate that these differential effects on cell growth were due to the expression of

mutants in the stable transfectants, cell lysates were examined for the expression of transgenes

by western blot analysis with myc-tag specific antibodies (Fig. 2C). Although all mutants

expressed in the transfectants, Wildtype TR and ∆-ID expressed to a lesser extent. In fact, the

expression of ∆-ID was lost with further passages of the transfectants (data not shown),

indicating its strong anti-cellular effects. To demonstrate a functional relationship between cell

death and the mutant expression, the stable transfectants were exposed to IFN/RA combination

(IFN/RA) and then stained with FITC-labeled annexin-V, a maker for apoptotic death (35). A

higher FITC positive staining indicates higher apoptosis. FITC positive cells were quantified

using flow cytometry (Fig. 2D). The TR and ID transfectants exhibited a significantly higher

sensitivity to IFN/RA induced cell death compared to the other mutants. ∆-ID expressing cells

became FITC 2 -2.5 fold higher than TR expressing ones. The FAD mutant acted as an inhibitor

of apoptosis, because cells expressing it were less FITC-positive, compared to the vector

expressing ones. Together these data indicate that ID of TR attenuates its pro-apoptotic effects.

Expression of ∆∆ -ID has no effect on endogenous TR and p53: To rule out a

possibility that the expression of TR or ∆-ID somehow altered the endogenous TR levels to

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mediate these differential effects, we next determined the levels of endogenous TR protein levels

by western blotting with antibodies specific for TR. Since only ∆-ID exhibited hyper death

stimulatory effects, we have selected it for further studies and compared its effects to full length

TR. As shown in Fig.3A neither the ∆-ID nor wildtype TR had an effect on the endogenous TR,

since its level was comparable between the vector and mutant transfected cells. In wildtype TR

transfected cells, a slow migrating band above the TR band was detected. It corresponds to the

TR protein derived from the transgene and migrates slower owing to the presence of an epitope

tag. This band is absent in the vector or ∆-ID expressing cells. Since the TR antibody was

directed against a peptide in the C-terminus, the ∆∆ -ID could not be detected.

We have shown earlier that the cell death effects of TR were in part due to its ability to

modulate tumor suppressor p53 dependent responses (25-27). Therefore, we examined the

possibility that a rise in endogenous p53 levels of mutant transfectants relative to vector

expressing cells occurred. Expression of ∆-ID or wildtype TR did not significantly affect p53

levels as revealed by a western blot analysis with an antibody that specifically detects a wildtype

p53 protein (Fig. 3B).

Effect of IFN/RA combination on p53 regulated gene expression: Earlier, we

have reported that TR modulates p53 dependent cell death via an upregulation of gene

expression (27). To test the influence of TR mutants on p53 stimulated gene expression we have

performed the following experiments. First we wanted to know whether ∆-ID induces the

expression a reporter gene driven by p53 response element. MCF-7 cells were transfected with a

p53RE-Luc reporter. Along with the reporter pCMV-flag, wildtype f -TR or f -∆-ID mutant were

cotransfected (Fig. 4A). Since flag tag is shorter and yielded a slightly better expression in

transient assays, we have used the flag tagged mutants for the following studies. Nevertheless,

the myc-tagged mutants exhibited similar properties like the flag-tagged ones (data not shown).

Following transfection cells were treated with IFN/RA and luciferase activity was measured.

IFN/RA induced luciferase expression in the vector transfectants. In TR transfectants basal

luciferase activity was elevated and it was further strongly induced by IFN/RA. ∆-ID co-

expression strongly enhanced and it was only slightly but significantly stimulated further by

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IFN/RA. ∆-ID induced the luciferase expression slightly higher than the IFN/RA treated, TR co-

expressed control. Previously we have shown that Bax, a p53 responsive mRNA and its protein

are induced in the presence of wildtype TR and inhibited in the presence of a catalytically

inactive mutant. Since p53 -Luc used in this experiment contained a synthetic promoter, we next

explored whether ∆-ID exerted a similar effect on a native promoter. A luciferase reporter driven

by human Bax has been shown to respond p53 (33,36). Therefore, we have employed it in the

next experiment. Fig.4B shows the data obtained. ∆-ID constitutively activated this promoter in a

manner similar to p53-Luc. TR on the other hand required treatment with IFN/RA to exert a

similar effect. Thus, a synthetic and the native promoter respond to ∆-ID similarly.

Previously we have shown that in the presence of a wildtype TR IFN/RA treatment

enhances the DNA binding of p53 and a catalytically inactive TR blocks it. Therefore, we

examined the DNA binding of p53 in cells stably expressing vector, wildtype TR and ∆-ID. Our

previous studies have shown that exposure of cells to IFN/RA for 24-28h, a time when

significant apoptosis can be detected is optimal for detecting p53 binding by EMSA, without

causing a rise in p53 levels (25-27). While an IFN/RA dependent induction of p53 binding

occurred in the TR expressing cells, a constitutive binding of p53 to the response element was

observed in cells expressing ∆-ID (Fig. 4C). It was slightly enhanced by IFN/RA. This

observation is consistent with the luciferase expression data. This band was competed out by a

cold p53binding element. We have shown earlier that this band is not competed out by a mutant

p53 oligonucleotide and it is supershifted by a polyclonal antibody against p53 (25-27).

p53 is necessary for a hyper stimulating effect of ∆∆ -ID on gene expression: A

critical role for p53 in TR regulated cell death effects was examined using DLD human colon

carcinoma cells, which lacked the endogenous p53 (37). Re-introduction of p53 causes death of

these cells (37). Introduction of a p53-Luc reporter along with an empty vector did not cause an

upregulation of luciferase activity. ∆-ID alone had no effect on luciferase expression. In the

wildtype p53 transfected cells luciferase expression was stimulated 3.5-4.0 fold over the vector-

transfected cells. It was induced further significantly enhanced in the presence of ∆-ID.

Furthermore, the same reporter was not induced in cells transfected with a mutant p53 and

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cotransfection of D-ID had no effect on (Fig. 5A). These data show that p53 is obligatory for an

inductive effect of ∆-ID on the luciferase reporter.

Human breast carcinoma cell line MCF-7 carries a wildtype p53 allele (38). The

endogenous p53 can be inactivated by targeting it to ubiquitin dependent proteolysis, upon stable

expression of the human papilloma viral (HPV)-E6 gene (29,30). Such epigenetic

downregulation confers a p53 null property to MCF-7-E6 cell line. The control cell line MCF-7

Neo carries an empty expression vector. Both cell lines were transfected with a p53RE-Luc

reporter along with empty vector, TR or ∆-ID construct. As shown in Fig.5B, transfection of

wildtype TR caused an elevation of luciferase gene expression in MCF7-Neo cells. ∆-ID also

induced luciferase expression, which was significantly higher than the wildtype TR. p53

dependent gene expression was induced by IFN/RA treatment in the vector-transfected cells. In

the presence of wildtype TR, IFN/RA further induced luciferase activity strongly. IFN/RA

caused only a marginal increase in luciferase expression in the ∆-ID transfected cells. Thus,

deletion of ID permits a constitutive activation of p53 dependent gene induction. The same

mutant when introduced into MCF7-E6 cells failed to promote luciferase expression. These

results show that p53 is critical for TR mediated gene induction.

Trx is required for hyperactivating p53 dependent gene expression: Since

mammalian TR exhibits a wide substrate range (23,24), we next examined whether the

hyperactivation of p53 dependent responses by ∆-ID was a result of its shift from the use of its

native substrate Trx1. Therefore, we next determined whether the ∆-ID mutant activates p53

dependent gene expression in MCF-7 cells stably expressing a mutant Trx, which lacks the

critical cysteines (at positions 32 and 35) for its redox function (25). For this purpose we have

employed three MCF-7 cell lines each stably expressing the vector (V), wildtype Trx1 (W) and

mutant Trx1 (M). Co-transfection of pCMV2 -flag with p53-Luc had no effect on luciferase

activity in the V, W and M cell lines (Fig. 6). However, co-transfection of wildtype TR elevated

the basal expression of p53-luc in V cells, which was further stimulated in W cells. However, TR

failed to augment p53 dependent gene expression in M cells. A similar pattern of gene regulation

was obtained with ∆-ID mutant in V, W and M cells. The only major difference between the ∆-

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ID and TR is that ∆-ID enhanced the gene expression to a higher level than TR. Thus, Trx is

required for ∆-ID’s stimulatory effect on p53 inducible expression.

Minimal region of TR protein required for stimulating p53 dependent gene

expression: Based on the above results we next determined the minimal region of ∆-ID

required for the stimulating p53 dependent gene expression. Serial deletion mutants each lacking

specific number of aminoacids from the C-terminus of ∆-ID were generated using PCR. These

mutants: ∆-ID80, ∆-ID70, ∆-ID60, ∆-ID50 and ∆-ID30, which lacked 34, 44, 54,64 and 95

aminoacids, respectively, were expressed as N-terminal flag tagged proteins using pCMV-flag.

These mutants yielded 23.6, 22.6, 21.6, 20.6, and 17.1 peptides, respectively. A western blot

analysis of transfected cell extracts with flag-tag specific antibodies showed the expression of

these mutants (Fig.7A). All mutants expressed equivalently. We next determined the effect of

these mutants on p53 dependent gene expression (Fig.7B). The ∆-ID80 mutant was better than ∆-

ID70 at augmenting the reporter gene expression constitutively, although ∆-ID70 still retained a

significant amount of stimulatory effect. The other mutants lost their stimulatory effects on p53

dependent gene expression. Although a slight stimulatory effect of IFN/RA was found on the ∆-

ID80 mutant, it was lost with the ∆-ID70. These data suggest that a domain between ∆-ID80 and

∆-ID60 regulates the constitutive stimulatory effect on p53 regulated gene expression.

Residues critical for the stimulatory effect on p53 dependent transcription: In

the next set of experiments we determined the residues critical for the hyperstimulatory effect of

∆-ID on p53 dependent gene expression. Based on the fact that ∆-ID80 exhibits a strong

constitutive effect on p53 dependent gene expression (Fig. 7B), we engineered new point

mutants that lack specific residues. Since the ∆-ID-70 mutant has significant stimulatory effect,

we reasoned that residues might lie within it. Primary sequence analysis of this region revealed a

NADPH binding domain. There are potential residues that can be phosphorylated in this region.

These include a serine at 199, a threonine at 193, two tyrosine residues at 187 and 200. Two of

these are present within the NADPH binding motif. While mutating tyrosine 187, we have also

converted the adjacent cysteine residue at 189 into an alanine, simultaneously. We have, thus,

generated 3 new mutants: 1) Y187F & C189A; 2) T193A; and 3) S199A & Y200F. These

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mutants were cloned into pCMV-flag and their expression was verified by western blot analysis

of the transfected cell extracts (Fig. 8A). The mutants were co-transfected along with a p53-

luciferase reporter and analyzed for their stimulatory effect on the promoter. Mutants Y187F &

C189A; and T193A completely lost their stimulatory effect on p53 dependent gene expression.

However, mutant S199A & Y200F retained its stimulatory effect, comparable to that of ∆-ID80

(Fig. 8B). Lastly, single mutants of Y187F and C189A were activated the p53 dependent gene

expression like WID indicating that both amino acids play a crucial in the regulation of p53

dependent gene expression.

The influence of point mutants on p53 activation was analyzed by EMSA. Stable cells

lines expressing flag-tagged mutants were generated (see below in Fig. 9). Four cell lines each

expressing ∆-ID; Y187F & C189A; T193A; and S199A & Y200F were utilized for EMSA (Fig.

8C). While ∆-ID and the S199A & Y200F mutant caused an elevation of p53 binding to the

response element, the Y187F & C189A and T193A mutants did not. IFN/RA did not cause an

activation of p53 in cells expressing the Y187F & C189A and T193A mutants. IFN/RA had a

slight stimulatory e ffect on ∆-ID and the S199A & Y200F stimulated p53 binding to its response

element. These data are consistent with the luciferase expression data.

To examine the role of these mutants in IFN/RA induced cytotoxicity, stable transfectants

expressing the flag tagged mutants were plated and selected with IFN/RA and G418 (0.5 mg/ml)

fro 3 weeks. The Y187F & C189A; and T193A mutants resisted IFN/RA unlike TR, ∆-ID, and

S199A & Y200F which were killed by the combination (Fig. 9A). This property is consistent

with their respective p53 augmenting functions. Expression of these mutants in the cells was

confirmed by a western blot analysis with flag-tag specific antibodies (Fig. 9B and 9C).

However, the expression of S199A & Y200F was lost over several passages, indicating its

growth inhibitory effect.

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Discussion:

Thioredoxin (Trx), a ubiquitous redox protein, regulates a wide-array of cellular activities

including growth, transcription and immune responses (23). Its redox status is controlled by a

cytosolic enzyme thioredoxin reductase (TR). In mammals 3 different TR genes, TR1, TR2 and

TR3, which express in an organelle and tissue specific manner, have been identified to date

(39,40). The founding member of this family, TR1, is expressed ubiquitously (23). Physiologic

roles of the new members of TR family are unclear at present. Mammalian TR has broader

substrate specificity than its prokaryotic homologues (23). For example, it can reduce unrelated

compounds such as selenite, alloxan, 5,5’ dithiobis(2-nitorbenzoic acid), vitamin-K,

selenocysteine, selinodiglutathione and S-nitrosoglutathione, in addition to Trx. Since TR

modifies its substrates extremely rapidly and no stable prosthetic groups are involved in this

process (23), it has been technically difficult to define its intracellular targets. Trx is also

implicated in maintaining the redox status of transcription factors, AP1, PEBP2, NF-κB and

HIF1α (28,41-44).

Based on chemical inhibition and other correlative data Trx1 is implicated in cell growth

promotion in some cell types (24). However, it is not a universal phenomenon. Several studies

showed that Trx and TR participate in growth inhibitory actions. The Trx homologue of

Drosophila, deadhead, is essential for female meiosis and early embryonic development (45) but

not for DNA synthesis in vivo (46). Deletion of TR1 in Drosophila affects survival (47). It codes

for a cytoplasmic and a mitochondrial isoform both of which are necessary for the viability of the

fly. Similarly Trx1 null mouse embryos do not implant at all (48). Thioredoxin inhibits DNA

synthesis in the fertilized Xenopus eggs (49). In yeast, deletion of Trx gene causes an increase in

the frequency of mitotic cell cycles (50). TR is necessary for p53-dependent growth suppression

in fission and budding yeasts (51,52). Inhibition of Trx1 causes resistance to IFN-γ induced

apoptosis (53) and Trx producing hepatomas grow slow (54). Using a genetic tool we have

shown earlier that TR1 is critical for the cell death controlled by IFN/RA combination (22,55).

We and others have shown that tumor suppressor p53, caspases-8 and –3 are also regulated by

TR:Trx (25-27,56,57).

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Tumor suppressor p53 inhibits cell growth either by aborting the division cycle or by

inducing death (58); and is frequently inactivated gene in several human cancers. Its activity is

controlled by several post-translational mechanisms such as stability, phosphorylation by protein

kinases, ubiquitination, SUMOylation, acetylation and redox factors (59). We have reported

earlier that the IFN/RA combination promotes p53 dependent gene expression and cell death

using TR1 and Trx1 (27) without elevating the levels of p53 protein. p53 has two cysteines

(residues 242 and 176), which along with two histidines coordinate Zn2+ for DNA binding

(60,61). Oxidation of these cysteines ablates its transcriptional activity (60,62). Wild type human

p53, but not a cysteine mutant, inhibits growth in yeast (63,64). Redox control of p53 activity is

further substantiated by studies that showed tms1, a dehydrogenase, suppresses p53 induced

growth arrest (65). Lastly, studies using chimeric p53 proteins in yeast have revealed that in

addition to t he DNA binding site, the transactivation domain of p53 is also subject TR dependent

redox regulation (66). Retinoids are known to activate oxidative stress through an increased

synthesis of reactive oxygen species (67-70). Presence of ROS is a signal for p53 activation (58).

IFN/RA treatment induces TR and Trx in cell undergoing apoptosis (22,26). p53 is kept in a

reduced form by redox factor-1 (Ref-1), a down stream effector of TR:Trx system. Indeed, recent

studies have shown that Ref-1 promotes p53 dependent gene expression and cell death (33). Ref-

1 has been suggested to prevent the oxidation of cysteine residues of p53. Trx1 augments Ref-1

dependent gene expression through p53 (57). More importantly, the failure of ∆-ID to promote

gene expression in the absence of p53 (Fig. 5) clearly indicates an obligatory role for p53 in this

process. Since ∆-ID fails to i nduce the p53 responsive reporter in cells expressing a mutant Trx,

its effects are Trx-dependent (Fig. 5).

The present observations raise a question: what is the role of ID? Mammalian TR is a

dimeric selenoprotein. Mutational analyses have revealed that the mammalian TR has two redox

centers: one at the N-terminus in the FAD binding domain and the other at its C-terminus.

Phylogenetically well conserved of these, N-terminal redox center, is constituted by cysteines

residues at 59 and 64. A selenocysteine at the C-terminus forms the C-terminal redox center. In

an unusual manner, one of the two in frame stop codons at the 3’ end of the ORF in conjunction

with a stem-loop structure formed by a selenocystine incorporation sequence of the 3’

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untranslated region of TR mRNA is proposed to act as an acceptor for selenocysteinyl tRNA

leading to a co-translational incorporation of selenocysteine into the mature protein (24,71).

Recent studies showed that bovine and rat TRs lacking this selenocysteine have an extremely

reduced kcat value in vitro (72,73). However, this enzyme did not completely lose its enzymatic

activi ty. Furthermore, depending on the substrate used for the assay, its activity is normal as long

as the N-terminal primary redox center is retained (73). In fact, incubation of the mutant derived

protein with selenocysteine restored the activity dramatically in vitro (72). These data suggest

that selenocysteine in trans can restore the enzyme activity. However, a low occurrence of free

selenocysteine (74) suggests that such modulation is a less frequent event in vivo. It is possible

that stress conditions such as apoptosis alter the physiologic availability of selenocysteine. Such

selenocysteine may be derived from an apoptotic degradation of other selenoproteins or its

biosynthesis. Atleast, 10-12 proteins that contain selenocysteine (75) have been identified to

date, degradation of which may provide free selenocysteine.

That yeast TR, which lacks a selenocysteine (76,77), can promote p53 dependent cell

growth arrest (51,52) and a truncated human TR lacking its ID also promotes p53 dependent

transcriptional response (this study) are consistent with the proposition that TR-stimulated p53-

mediated responses are selenocysteine-independent. Similarly, Drosophila and plant TRs do not

require selenium for their function (47,78). In fact, plant TR behaves like a prokaryotic TR (78).

Furthermore, the prokaryotic TRs do not reduce other substrates, like the mammalian TR. Recent

crystallographic data on rat TR (79) have shown that the enzyme forms a head-to-tail dimer, with

the N-terminal redox center buried inside. Reducing power is first transferred from NADPH to

the N-terminal redox center. The C-terminus of one subunit is inserted to the charged cleft (N-

terminal redox center) of the other subunit to tap electrons from the active site to selenocysteine.

The reduced selenocysteine then donates electrons to Trx/other substrates at the surface of TR

dimer, after emerging from the catalytic cleft. In this model, the ID extends like a robotic arm to

transfer electrons between the enzyme and its various substrates. In the case of prokaryotic TR,

which also acts as a dimer, after the receipt of electrons at the redox center from NADPH, the

NADPH binding domain undergoes a 66o rotation for providing an access to oxidized Trx to the

redox center (23,24). ∆-ID may behave like prokaryotic, yeast, and plant TRs with strict Trx

reductive properties. This would be sufficient for activating p53. Thus, it would appear that a

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selenocysteine at the C-terminus has evolved to enhance catalysis and broaden substrate r ange

and is an optional accessory. Although an augmentation of TR activity by selenium has been

reported in mammalian cells, these studies used a supra-nutritional concentration of selenium

and are not physiologically relevant. In fact, an inverse correlation between selenium and TR

activity has also been reported (80). Lastly, selenium metabolites can activate cell cycle arrest in

the absence of p53 and DNA damage (80), indicating the existence of a separate mechanism of

action. In the light of these data, we suggest that selenium/selenocysteine plays a limited role in

TR mediated growth suppressive pathways in vivo, but are required for other redox reactions

during normal growth.

Alternatively, TR bearing alkylated C-terminal Selenocysteine and Cysteine residues

exhibits 30-fold higher NADPH oxidase activity compared to the wildtype enzyme, and is

capable of producing superoxides (81). These superoxides can oxidize intracellular environment,

thus tilting the balance towards p53 activation and cell death. Consistent with this suggestion,

deletion of NADPH binding domain (mutants ∆-ID60 and ∆-ID50) prevents the stimulatory

effects of TR on p53 dependent gene expression (Fig. 7). It is interesting to note that the

mutation of cysteine residue at 189 depletes the p53-augmenting function of ∆-ID (Fig. 8). This

suggests that Trx transiently interacts with this site during enzymatic modification, since Trx1 is

still necessary for promoting p53-dependent gene expression. The threonine and tyrosine

residues may undergo post-translational modifications, which in turn contribute to full activity of

∆-ID. Thus, ID, which is unique to mammalian TR, appears act as a regulatory switch in cell

growth control. Its presence attenuates the growth inhibitory effect of TR and its removal

promotes cell death. Since prokaryotic/unicellular organisms do not undergo apoptosis and

primary role of TR is only to maintain the redox functions they have a much simpler modular

structure. Because TR and Trx can promote pro- and anti- growth processes depending on the

physiologic status of cells and mammalian TR has expanded physiologic roles, evolution of a

regulatory switch (ID) is critical for preventing an inadvertent operation of these divergent

processes. Thus, ID may act as a decision switch to mediate such “Yin-Yang” reactions in vivo.

In the meanwhile, one potential use for the ∆-ID mutant will be in gene therapy along with

wildtype p53 in p53 null tumors.

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Acknowledgments: These studies are supported by the National Cancer Institute grants CA

78282 and CA 71401 to DVK. The authors thank Peter Gutierrez for helpful discussions.

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Legends to Figures:

Figure 1: Expression of TR mutants. Mutants were generated using PCR as described in

materials and methods section. Panel A: diagrammatic representation of the structures of TR

mutants. Panels and B show the western blot (WB) analyses. MCF-7cells were transiently

transfected with the indicated plasmids (1µg) and equal amounts of lysates were separated on a

10% SDS-PAGE and western blotted. The blots were probed with indicated antibodies.

Figure 2: Effects of TR mutants on cell growth. (A) Effect of TR mutants on G418r colony

formation. MCF-7 cells were transfected with equimolar amounts of pCXN2-myc vector

expressing various mutants After 3 weeks of selection with G418 (1 mg/ml) in growth medium,

surviving colonies were counted. Each bar represents mean ± SE of triplicates. (B) Effects of TR

mutants on cell growth. Equal number of cells (2000/well), stably transfected with various TR

mutants, were plated and cell growth was monitored after 5 days using the sulforhodamine B

binding assay (34). Each bar represents mean ± SE of 8 replicates. Cell growth was monitored

quantified by measuring the absorbance of bound dye at 575 nm. (C) Expression of TR mutants

after stable transfection in MCF-7 cells. Total protein (65 µg) from the indicated cell lines was

western blotted and probed with anti-myc antibody. This blot was stripped and probed (shown

below) with actin specific antibodies. (D) Cells treated with IFN/RA were stained with annexin-

V as described under materials and methods. Percentage of annexin-V positive cells was

quantified using FACS analysis. Each bar represents mean ± SE of triplicates.

Figure 3: Equal amount of total cell lysate (85 µg) from the indicated cell lines were

immunoblotted and analyzed with s pecific antibodies. (A) Expression of endogenous TR protein.

Arrowhead indicates the position of transgene derived TR. This antibody does not recognize

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protein derived from ∆-ID. This blot was stripped and probed with anti-actin antibodies (shown

below). (B) Expression of endogenous p53 protein. WB: western blot.

Figure 4: Effect of IFN/RA on p53 dependent gene expression. (A) MCF-7 cells were treated

with IFN/RA after transfecting with a p53-Luc, CMV-β-galactosidase, and TR mutants. Cell

extracts were measured for luciferase and β-galactosidase activity. Luciferase activity was

normalized to the β-galactosidase activity. Each bar represents mean ± SE of triplicates. A plus

(+) sign indicates treatment with IFN-β (500 U/ml) and RA (1 µM) for 16h. (B) Effect of

IFN/RA on the Bax promoter. MCF-7 cells were transfected with Bax-Luc and CMV-β-

galactosidase plasmids. Cells were stimulated with IFN/RA where indicated with a plus sign. (C)

Effect of TR mutants on p53 binding to DNA. Cell extracts from IFN/RA stimulated cells (24h)

were incubated with a 32P-labeled oligonucleotide bearing the consensus p53 binding site. Plus

and minus signs indicate no treatment and IFN/RA treatment, respectively. Where indicated 50X

cold: the D-ID cell extract was incubated with an excess (50X) unlabeled oligonucleotide, prior

to use in EMSA. Thirty micrograms of nuclear extract was used in this experiment.

Figure 5: p53 is required for the gene stimulatory effect of ∆-ID. In panel A, human DLD colon

carcinoma cell line (p53 null) was transfected with the indicated plasmids along with p53-Luc

and CMV-β-galactosidase reporters. A plus sign indicates the presence of the indicated plasmid

in the transfection mixture. MCF-7 neo and MCF-7E6 cells were transfected with p53RE-

Luciferase in the presence of various indicated plasmids. Total amount of DNA transfected into

the cells was kept constant (1.0 µg) by adding the pCMV-flag vector, where required.

Figure 6: Trx1 is required for the hyperstimulation of p53 dependent gene expression.

MCF-7 cells stably expressing control vector (V), Wildtype (W) and redox inactive Trx1 (M)

were transfected with TR mutants along with p53-Luc and CMV-β-galactosidase reporters.

Luciferase activity was quantified as described in figure 4. Plus sign indicates the presence of

that specific plasmid in transfection mixture.

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Figure 7: Minimal region of ∆-ID required for its hyperstimulatory effects on p53 responsive

gene expression. (A) Expression of mutants in MCF-7 cells. Cells in 6 well dishes were

transfected with 0.5 µg of indicated plasmids and whole cell lysates were prepared. A

comparable quantity of protein (50µg) from each sample was employed for western blot analysis

using flag specific antibodies. (B) Effect of the mutants shown in panel A on p53-dependnent

luciferase reporter. Indicated plasmids were co-transfected with p53-Luc and CMV-β-

galactosidase reporters and luciferase expression was analyzed. A plus (+) sign indicates

treatment with IFN-β (500 U/ml) and RA (1 µM) for 16h.

Figure 8: Residues required for the hyperstimulatory effect of ∆-ID on p53 mediated gene

expression. Panel A shows the expression of the point mutants indicated above it. This

experiment is similar to Fig. 7A. Panel B shows the effect of point mutants on p53 dependent

gene expression. Transfection and reporter gene analysis was similar to fig. 7B. (C) EMSA for

p53 activation. Stable cell lines expressing flag-tagged TR mutants (indicated above the lanes)

were treated with IFN/RA for 30h and EMSA was performed. A plus (+) sign indicates treatment

with IFN-β (500 U/ml) and RA (1 µM). Location of p53 band is indicated.

Figure 9: Effect of IFN/RA on cells expressing TR mutants. (A): Cells stably expressing flag-

tagged TR mutants were selected for 3 weeks with IFN-β (500 U/ml) and RA (1 µM) for 3

weeks. Cells were then stained with sulforhodamine B to visualize surviving cells. (B) & (C):

Expression of TR mutants in the stable transfectants. Extracts from the cells transfected with the

indicated plasmids (70µg) were employed for western blot analysis using anti-flag antibodies.

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pC

MV

-fla

g

f-T

R

f-∆∆

-FA

D

f-∆∆

-NB

D

f −−∆∆

-ID

Figure 1

WB:anti-flagWB:anti-myc

TR

∆∆-FAD

∆∆-NBD

∆∆-ID

[A]

[B]

pC

XN

2-m

yc

TR

-myc

∆∆-F

AD

-myc

∆∆-N

BD

-myc

∆∆-I

D-m

yc

[C]

FAD IDNBD

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0

0.2

0.6

0.4

Ve

cto

r

Wt

∆∆-F

AD

∆∆-N

BD

∆∆-I

D

Ab

sorb

ance

Ve

cto

r

Wt

∆∆-F

AD

∆∆-N

BD

∆∆-I

D

800

0

200

400

600

G1

48

rco

lon

ies

Ve

cto

r

TR ∆∆-F

AD

∆∆-N

BD

∆∆-I

D

actin

anti-myc

10

50

40

30

20

0

Ve

cto

r

Wt

∆∆-F

AD

∆∆-N

BD

∆∆-I

D

Pe

rce

nt

An

ne

xin

-VP

osi

tive

[A] [B]

[C][D]

Figure 2

WB: Anti-p53

Ve

cto

r

TR ∆∆-I

D

WB: Anti-actin

[B]

WB: Anti-TR

Ve

cto

r

TR

∆∆-I

D

E

[A]

Figure 3

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RLU

(1X

10

3)

0

8

4

6

2

IFN/RA - + - + - +

Vector TR ∆∆-ID

Figure 4

0

10

5.0

7.5

2.5

IFN/RA - + - + - +Vector TR ∆∆-ID

[A]

[B]

RLU

(1X

10

3)

IFN/RA - + - + - + - + - + - +

RLU

(1X

10

3)

8

4

6

2

0

Vector TR ∆∆-ID Vector TR ∆∆-ID

MCF-7 Neo MCF-7 E6

[B]

Figure 5

15

9

12

6

Vector + + - - - -p53-wt - - + + - -

3

0∆∆-ID - + - + - +

p53-mut - - - - + +

[A]

RLU

(1X

10

3)

Ve

cto

r

TR

∆∆-I

D

IFN/RA - + - + - + No

ex

tra

ct5

0X

co

ld

p53

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Figure 6

∆∆ID - - - - - - + + +

RLU

(1X

10

3)

12

3

9

0

6

pCMV-flag + + + - - - - - -TR - - - + + + - - -

Cell line with: V W M V W M V W M

∆∆-I

D-8

0

∆∆-I

D-7

0

∆∆-I

D-6

0

∆∆-I

D-5

0

∆∆-I

D-3

0

∆∆-I

D

Ve

cto

r

10

20

30

4060

[A]

RLU

(1X

10

3)

10

4

8

6

2

0IFN/RA - + - + - + - + - + - + - +

Vector ∆∆-ID ∆∆-ID80

∆∆-ID70

∆∆-ID60

∆∆-ID50

∆∆-ID30

[B]

Figure 7

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∆∆-ID80

Vector

8

4

6

2

0

10

Y187F C189A

T193A S199A Y200F

RLU

(1X

10

3)

Y1

87

F C

18

9A

T1

93

A

S1

99

A

Y2

00

F

Ve

cto

r

Figure 8

WB: anti-flag

[A]

[B]

IFN/RA - + - + - + - + - +

IFN/RA - - + - + - + - +

[C]

Y1

87

F C

18

9A

T1

93

A

S1

99

A

Y2

00

F

∆∆-I

D

p53

No

ne

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Y1

87

F C

18

9A

T1

93

A

S1

99

A

Y2

00

F

Ve

cto

rVe

cto

r

TR ∆∆-I

D[B] [C]

Y187F C189A

Vector TR ∆∆-ID

[A]

T193A S199A Y200F

Figure 9

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Table 1: Primers used in this study

Primers for FLAG tagged constructs:

Flag primer1: 5’-CGGAATTCCATGAACGGCCCTGAAGAT –3’Flag primer2: 5’-GGCCATGGTCAGCAGCCAGCCTGGAGGAT-3’Flag primer 3: 5’-CCGGAATTCGCAATTTATTGGTCCTCAC-3’Flag primer4: 5’-GGCCATGGTCAATTGGTCTGTTCTTCATC-3’

∆∆-ID-80 R-primer : 5’-CGG GGT ACC TCA GAC GTT TAA ACC AAT ACC-3’∆∆-ID-70 R-primer: 5’-CGG GGT ACC TCA AGC GCA CTC CAA AGC GAC-3’∆∆-ID-60 R-primer : 5’-CGG GGT ACC TCA AAC AAC CAG GGT CTT ACC-3’∆∆-ID-50 R-primer : 5’-CGG GGT ACC TCA CAA GGA GAA AAG ATC ATC-3’∆∆-ID-30 R-primer : 5’-CGG GGT ACC TCA TCT CTC TGC TGA ATA AAT-3’

Y187F & C189A Forward primer: 5’-TTC TCC TTG CCT TTT GCT CCG GGT AAG ACC-3’Reverse primer: 5’-GGT CTT ACC CGG AGC AAA AGG CAA GGA GAA-3’

T193A - Forward primer: 5’-TGC CCG GGT AAG GCT CTG GTT GTT GGA GCA-3’Reverse primer: 5’-TGC TCC AAC AAC CAG AGC CTT ACC CGG GCA-3’

S199A & Y200F Forward primer: 5’-GTT GTT GGA GCA GCT TTT GTC GCT TTG GAG-3’Reverse primer: 5’-CTC CAA AGC GAC AAA AGC TGC TCC AAC AAC-3’

Y187F: Forward primer:5’-TTC TCC TTG CCT TTT TGC CCG GGT AAG ACC-3’Reverse primer: 5’-GGT CTT ACC CGG GCA AAA AGG CAA GGA GAA-3’

C189A: Forward primer: 5’-TTC TCC TTG CCT TAC GCA CCG GGT AAG ACC-3’Reverse primer: 5’-GGT CTT ACC CGG TGC GTA AGG CAA GGA GAA-3’

Primers for expressing myc-tagged mutants:

Myc-Primer 1: 5’-CGGAATTCGCCACCATGAACGGCCCTGAAGAT-3’Myc-Primer 2: 5’-GGCCATGGGCAGCCAGCCTGGAGGAT-3’Myc-Primer 3: 5’-CCGGAATTCCAATTTATTGGTCCTCAC-3’Myc-Primer 4: 5’-GGCCATGGATTGGTCTGTTCTTCATC-3’∆∆-NBD primer1: 5’-CGCGGATCCATAAGCATTCTCATAGAC-3’∆∆-NBD primer 2: 5’-CGCGGATCCGTGCCTTACATCTATGCCATT-3’

Mutated sites have been shown with bold face. All myc primers contained EcoRI and Kpn I at the 5’ and 3’ ends respectively. The 5’ and 3’ ends of flag primers bear EcoRI and BamHI sites respectively.

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Xinrong Ma, Junbo Hu, Daniel J. Lindner and Dhananjaya V. Kalvakolanuexpression and interferon and retinoic acid induced cell death

Mutational analysis of human thioredoxin reductase 1: Effects on p53 mediated gene

published online April 12, 2002J. Biol. Chem. 

  10.1074/jbc.M202286200Access the most updated version of this article at doi:

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