Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M303420200 on July 21, 2003

J. Biol. Chem., Vol. 278, Issue 40, 38325-38332, October 3, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/40/38325    most recent
M303420200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, M.
Right arrow Articles by May, W. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, M.
Right arrow Articles by May, W. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

A Novel Role for RAX, the Cellular Activator of PKR, in Synergistically Stimulating SV40 Large T Antigen-dependent Gene Expression*

Mingli Yang, Takahiko Ito {ddagger} and W. Stratford May §

From the University of Florida Shands Cancer Center and Department of Medicine, University of Florida, Gainesville, Florida 32610-0232

Received for publication, April 2, 2003 , and in revised form, June 30, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The double-stranded (ds) RNA-binding protein RAX was discovered as a stress-induced cellular activator of the dsRNA-dependent protein kinase (PKR), a key regulator of protein synthesis in response to viral infection and cellular stress. We now report a novel function of RAX, independent of PKR, to enhance SV40 promoter (origin)/enhancer-dependent gene expression. Several mammalian cell lines including COS-7, CV-1, and HeLa cells were tested. Results reveal that the SV40 large T antigen is required for RAX-mediated, synergistic enhancement of gene expression. RAX augments SV40 regulatory element-dependent DNA replication and transcription. The mechanism requires the SV40 enhancer, a viral transcriptional element that is necessary for efficient SV40 DNA replication in vivo. Mutational analysis reveals that the dsRNA-binding domains of RAX are required for the gene expression enhancing function. Thus, in addition to stimulating PKR activity, RAX can positively regulate both SV40 large T antigen-dependent DNA replication and transcription in a mechanism that may alter the interaction of the cellular factor(s) with the SV40 enhancer via the dsRNA-binding domains of RAX. This novel function of RAX may have implications for regulation of mammalian DNA replication and transcription because of the many similarities between the viral and cellular processes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SV40 is a small DNA virus that has served as a powerful model system for dissecting fundamental biological processes including, DNA replication, transcription, and neoplastic transformation (1–4). The SV40 genome consists of a 5.2-kb circular duplex DNA molecule with a 300-bp regulatory region that contains SV40 promoter (including the viral origin of DNA replication) and enhancer elements (2, 5). SV40 DNA replication and transcription occurs in the nucleus of the host cell where the SV40 genome is complexed with histones to form a nucleosomal structure (i.e. minichromosome) that is indistinguishable from cellular chromatin, indicating the similarities between viral and cellular DNA replication and transcription (2, 6, 7). The SV40 large T antigen (T-Ag)1 is a multifunctional viral protein with DNA helicase activity and transcriptional activity and plays an essential role in SV40 DNA replication and transcription (1–4). The T-Ag has been found to interact with a number of cellular proteins that regulate DNA replication and transcription (e.g. DNA polymerase {alpha}, human TFIIB-related factor, pRb, and p53) and to modify cellular regulatory processes that may efficiently promote viral gene expression and replication in permissive cells (1–4). However, the mechanisms by which SV40 T-Ag cooperates with the cellular proteins in vivo to facilitate viral DNA replication and transcription have not been fully elucidated.

The interferon-inducible double-stranded (ds) RNA-dependent protein kinase (PKR) is a major regulator of host antiviral defense and normal cellular protein synthesis (8–11). For example, viral infection leads to PKR activation with phosphorylation of its physiologic substrate, the {alpha} subunit of eukaryotic initiation factor-2 (eIF2{alpha}), that leads to inhibition of translation of mRNA (12). PKR also shuts down protein synthesis in uninfected cells following cellular stress responses that lead to inhibition of cell growth and apoptosis, including interleukin-3 growth factor withdrawal, serum deprivation, and treatment of cells with tumor necrosis factor-{alpha} and lipopolysaccharide (11, 13, 14). Recently, RAX and its human homologue, PACT, were independently discovered as the first cellular activators of PKR (15, 16). RAX/PACT is also a dsRNA-binding protein that directly binds and activates PKR following cellular stresses (such as interleukin-3 withdrawal, sodium arsenite, hydrogen peroxide, serum deprivation, or ceramide treatment) that lead to cell death (15–18). In addition, it is now clear that PKR is a key regulator of protein synthesis but may also regulate other fundamental cellular processes including cell proliferation, apoptosis, differentiation, and signal transduction (19–22). Findings reported here indicate that RAX has a novel, unexpected function to augment SV40 T-Ag-dependent DNA replication and transcription that is dependent on its dsRNA-binding domains but independent of PKR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—COS-7, CV-1, and HeLa cells were obtained from American Type Culture Collection and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. All transient transfection or co-transfection in these cells was performed using LipofectAMINE (Invitrogen).

Luciferase Reporter Constructs and Expression Constructs—pGL3-luciferase reporter vectors (including pGL3-promoter vector and pGL3-basic vector) and pRL-Renilla luciferase reporter vectors (including pRL-SV40 vector and pRL-CMV vector) were obtained from Promega. pGL3-promoter vector contains the SV40 early promoter element, whereas pGL3-basic vector lacks promoter or enhancer elements. pRL-SV40 vector and pRL-CMV vector contain the SV40 early promoter/enhancer elements and the human cytomegalovirus (CMV) early promoter/enhancer elements, respectively. pGL3-SV40, pGL3-CMV, and pGL3-RSV luciferase reporter vectors were also created by subcloning the promoter/enhancers of SV40, CMV, and Rous sarcoma virus (RSV) into the pGL3-basic vector, respectively. The SV40 promoter/enhancer sequence in the pGL3-SV40 and pRL-SV40 vectors contains the SV40 core origin of DNA replication and auxiliary elements including the 21-bp repeats and the 72-bp enhancer elements (23). pSV40 E-TAL luciferase reporter vector was created by subcloning the SV40 enhancer sequence from pRL-SV40 vector into pTAL-luciferase reporter vector (Clontech) that contains a TATA-like promoter (PTAL) region from the Herpes simplex virus-thymidine kinase promoter.

The hemagglutin (HA) epitope-tagged RAX cDNA (15) was subcloned into expression vectors pcDNA3 (Invitrogen) and pGC1, respectively. The pGC1 vector was derived from the pGL3-CMV vector by deleting the luciferase reporter gene cDNA. Expression plasmids of FLAG-tagged RAX and its mutants (such as K84A, K177A, {Delta}II and {Delta}I/II) were also created using expression vectors pIRESneo (Clontech) and pcDEF3 (15, 24), respectively. Site-directed mutagenesis experiments were performed using the Transformer Kit (Clontech). In K84A and K177A mutants the lysine codon was mutated to an alanine codon. {Delta}II and {Delta}I/II mutants were created by deleting the cDNA fragments coding amino acid residues 125–194 or residues 63–219 using the restriction enzymes followed by blunt-end ligation.

An expression construct of SV40 large T antigen was created by subcloning the T-Ag cDNA fragment of SV40 genome/pRB322 into the pIRESneo vector. A FLAG-tagged eIF2{alpha} cDNA was also subcloned into the pcDNA3 vector. The expression vectors pcDNA3 and pcDEF3 contain the SV40 promoter/enhancer elements that function as the neomycin promoter, whereas pGC1 and pIRESneo do not contain such SV40 regulatory elements.

Co-expression of RAX and eIF2{alpha}—Co-transfection of 0.5 µg of FLAG-RAX/pIRESneo (empty vector as a control) with 0.5 µg of FLAG-eIF2{alpha}/pcDNA3 into COS-7 cells was performed in 6-well plates. After 48 h cells were lysed in buffer A (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and protease inhibitor mixture (Sigma)). 50 µg of the lysate was loaded into a 10% SDS-PAGE gel followed by Western blot analysis using an anti-FLAG antibody (Roche Diagnostics) and the same blot was also stained by Fast Green dye.

Luciferase Assays—Co-transfection of 1 µg of HA-RAX/pGC1 (empty vector as a control) with 0.1 µg of pGL3-SV40 vector, pGL3-RSV vector, or pGL3-CMV vector into COS-7 cells was performed in 6-well plates. After 48 h cells were lysed and assayed for luciferase activity using the luciferase assay system (Promega). 0.1 µg of pGL3-SV40 luciferase vector was also co-transfected into CV-1 cells with a total of 1 µg of plasmids containing empty vector, RAX alone, T-Ag alone, or both RAX and T-Ag followed by luciferase assays. The plasmid combinations were 0.5 µg of pGC1 plus 0.5 µg of pIRESneo for the "empty vector," 0.5 µg of HA-RAX/pGC1 plus 0.5 µg of pIRESneo for the "RAX alone," 0.5 µg of pGC1 plus 0.5 µg of T-Ag/pIRESneo for the "T-Ag alone," and 0.5 µg of RAX/pGC1 plus 0.5 µg of T-Ag/pIRESneo for the "both RAX and T-Ag," respectively. Similar experiments were also carried out in HeLa cells. The total protein was measured for normalizing luciferase activity readings.

To rule out the possibility that the observed effect of RAX on luciferase activity might be an artifact of a particular RAX expression vector or luciferase reporter vector used, other RAX expression plasmids (including HA-RAX/pcDNA3 and Flag-RAX/pIRESneo) and other luciferase reporter vectors (including pRL-SV40 and pRL-CMV Renilla luciferase vectors as well as pGL3-control vector (Promega), which contains both SV40 promoter and enhancer elements and was alternatively used as a pGL3-SV40 vector) were also tested in the studies. Similar results were obtained and some examples are shown under "Results."

Treatment of the PKR inhibitor 2-aminopurine (2-AP) was performed (25). After the HA-RAX/pGC1 plasmid (empty vector as a control) was co-transfected with the pGL3-SV40 luciferase vector into COS-7 cells using the LipofectAMINE method, 2-AP was immediately added into the Dulbecco's modified Eagle's media to a final concentration of 10 mM. After 48 h cells were lysed and luciferase activity was assayed as described above.

Southern Blot Analysis—Co-transfection in COS-7 and CV-1 cells was carried out as described before. For plasmid isolation, cells were harvested and lysed in the plasmid lysis buffer (0.6% SDS, 10 mM EDTA). The lysate was added with NaCl to a final concentration of ~1 M and incubated on ice overnight. The lysate was then centrifuged for 15 min at 13 x g to pellet chromosome DNA. The plasmids in the supernatant were extracted by phenol/chloroform and then precipitated by ethanol. The purified plasmids were linearized by the restriction enzymes and then digested by DpnI (26) followed by Southern blot analysis using the luciferase cDNA insert or the FLAG-eIF2{alpha} cDNA insert as probe that was radiolabeled using the RadPrime DNA labeling system (Invitrogen).

Northern Blot Analysis—Co-transfection was performed as described above. Total RNAs were isolated using the Trizol method (Invitrogen) and probed with the radiolabeled luciferase cDNA insert.

[35S]Methionine Labeling and Immunoblotting—Co-transfection was performed as described above. After 48 h, proteins in COS-7 cells that transiently express HA-RAX (or empty vector as a control) were pulse-labeled with [35S]methionine for 60 min to assess protein synthesis (27). The cells were lysed in buffer B (10 mM HEPES, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 20 mM sodium fluoride, 20 mM sodium pyrophosphate, 20 mM {beta}-glycerophosphate, 20 mM sodium molybdate, 20 mM microcystin-LR and protease inhibitor mixture (Sigma)) followed by SDS-PAGE, Western transfer, and autoradiography. The same blot was also immunoblotted using antibodies against phospho-eIF2{alpha} (Cell Signaling Technology), eIF2{alpha} (15), HA (12CA5, Roche Diagnostics), and SV40 T-Ag (BD Pharmingen).

Poly(I)·Poly(C)-Agarose Beads Binding Assays—Expression plasmids of FLAG-tagged wild-type and mutant RAX as described earlier were transfected into COS-7 cells, respectively. After 48 h cells were lysed in buffer A. Aliquots (50 µg) of lysates were incubated with 20 µl of poly(I)·poly(C) or poly(C)-agarose beads (Amersham Biosciences) for 1.5hat4 °C. These beads were washed three times with buffer A before boiling in SDS-PAGE sample buffer. The eluted samples and 50 µg of the whole cell lysate (as input) were then loaded onto a 10% SDS-PAGE gel followed by immunoblotting using an anti-FLAG antibody.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RAX Selectively Stimulates SV40 Promoter/Enhancer-dependent Gene Expression—To investigate the mechanism(s) by which RAX may regulate PKR, COS-7 cells were co-transfected with plasmids containing FLAG-tagged RAX and FLAG-tagged eIF2{alpha}. Surprisingly, Western blot analysis revealed that FLAG-RAX could significantly enhance the expression of the FLAG-eIF2{alpha} plasmid that was created in a pcDNA3 vector (Invitrogen, Fig. 1A). Fast Green staining confirmed that expression of total cellular proteins was not affected (Fig. 1B). RAX-mediated augmentation of exogenous gene expression was unexpected because RAX was initially identified and characterized as a cellular activator of PKR that inhibits protein synthesis (15, 16). Because viral promoter/enhancers (such as those of SV40, CMV, and RSV) are commonly used to drive exogenous gene expression, we tested whether RAX could affect the transcriptional activity of these promoter/enhancer elements. A commercial pGL3-luciferase reporter vector (Promega) containing these viral promoter/enhancer elements was constructed and co-transfected into COS-7 cells along with a plasmid containing either an HA-tagged RAX or an empty vector. Luciferase expression results indicated that HA-RAX could significantly enhance SV40 promoter/enhancer-dependent gene expression (i.e. ~10-fold increase) but had only a minimal effect on gene expression driven by the CMV or RSV promoter/enhancers (i.e. ~1-fold increase, Fig. 2A). Western analysis demonstrates equal levels of HA-RAX or T-Ag expression (Fig. 2B).



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 1.
RAX-mediated enhancement of FLAG-eIF2{alpha} expression in COS-7 cells. FLAG-RAX/pIRESneo (empty vector as a control) was co-transfected with FLAG-eIF2{alpha}/pcDNA3 into COS-7 cells. At 48 h post-transfection, cells were lysed and 50 µg of the lysate was loaded into a 10% SDS-PAGE gel as described under "Materials and Methods." A, Western blot analysis using an anti-FLAG antibody. B, Fast Green staining. V, empty vector pIRESneo; RAX, FLAG-RAX/pIRESneo. Lanes 1–3 are triplicates for the vector, whereas lanes 4–6 represent triplicates for FLAG-RAX.

 


View larger version (22K):
[in this window]
[in a new window]
  		FIG. 2.
Effects of RAX on luciferase reporter gene expression in COS-7 cells. 1 µg of HA-RAX/pGC1 plasmid (empty vector as a control) was co-transfected into COS-7 cells with 0.1 µg of pGL3-luciferase reporter vector containing the promoter/enhancer of SV40, RSV, or CMV (pGL3-SV40, pGL3-RSV, or pGL3-CMV). After 48 h cells were lysed and assayed for luciferase activity as described under "Materials and Methods." A, relative luciferase activity. Luciferase expression is displayed relative to that of the vector. V, empty vector pGC1; RAX, HA-RAX/pGC1. B, Western blot analysis using antibodies of anti-HA and anti-T-Ag. Lanes 1 and 4, pGL3-SV40; lanes 2 and 5, pGL3-RSV; lanes 3 and 6, pGL3-CMV.

 

SV40 Large T Antigen Is Required for RAX-mediated Enhancement of Luciferase Reporter Gene Expression—COS-7 cells were derived from CV-1 monkey kidney cells following transformation with a replication origin-defective SV40 mutant that encodes the wild type T-Ag (28). Abundant expression of T-Ag occurs in COS-7 but not CV-1 cells (Figs. 2B and 3B). To test whether T-Ag expression was required for the RAX-mediated enhancement of luciferase gene expression, CV-1 cells were also co-transfected with a pGL3 SV40-luciferase vector and the plasmids containing HA-tagged RAX and/or T-Ag. Results indicated that while expression of HA-RAX alone had essentially no effect on luciferase gene expression, co-expression with the T-Ag can synergistically enhance the activity by over 45-fold (Fig. 3A). Because both HA-RAX and T-Ag were expressed at similar levels in CV-1 cells when transfected individually or together, variable expression levels could not account for HA-RAX-mediated enhancement of luciferase expression (Fig. 3B). Furthermore, in addition to monkey COS-7 and CV-1 cells that are naturally SV40 permissive (28), RAX was also demonstrated to synergize with the T-Ag to stimulate SV40 promoter/enhancer-dependent luciferase gene expression in human HeLa cells (Fig. 4A). These findings clearly demonstrate that T-Ag expression is necessary for the synergistic enhancing effect of RAX on luciferase gene expression.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 3.
Role of T-Ag in RAX-mediated enhancement of gene expression. 0.1 µg of the pGL3-SV40 luciferase reporter vector was co-transfected into CV-1 cells with 1 µg of plasmids containing empty vector (V), HA-RAX alone, T-Ag alone, or both HA-RAX and T-Ag. After 48 h cells were lysed and assayed for luciferase activity as described under "Materials and Methods." A, relative luciferase activity. B, Western blot analysis using antibodies of anti-HA and anti-T-Ag. Lane 1, control, the lysate from COS-7 cells that was transfected with HA-RAX.

 


View larger version (13K):
[in this window]
[in a new window]
  		FIG. 4.
A, RAX-mediated enhancement of luciferase reporter gene expression in HeLa cells. pGL3-SV40 luciferase reporter vector was co-transfected into HeLa cells with plasmids containing RAX and/or T-Ag followed by luciferase assays similarly as described in the legend to Fig. 3. B, effects of RAX on Renilla luciferase reporter gene expression in COS-7 cells. pRL-SV40 and pRL-CMV Renilla luciferase reporter vectors were used to repeat luciferase assays as described in the legend to Fig. 2.

 

To rule out a nonspecific effect from the pGL3-Firefly luciferase reporter gene vector that might account for any differential reporter gene expression observed, a second luciferase reporter gene from another species was also tested. The pRL Renilla luciferase reporter vector (Promega) that contains either SV40 or CMV promoter/enhancer elements was expressed in COS-7 cells (Fig. 4B). Results confirm that HA-RAX exerts a selective and specific effect that was not dependent on the luciferase gene because expression of the two different luciferase enzymes were both synergistically enhanced. These data rule out a nonspecific, species-specific enhancement of gene expression. Therefore, it is concluded that RAX-mediated enhancement of luciferase gene expression is specific for the SV40 promoter/enhancer elements and independent of the plasmid or reporter genes employed.

RAX-mediated Enhancement of Gene Expression Occurs at the Level of Both DNA Replication and mRNA Expression—The wild-type SV40 origin of DNA replication is contained in both the pGL3- and pRL-luciferase reporter vectors. Plasmids containing this origin can replicate in cells expressing the T-Ag, such as COS-7 cells (28). Therefore, we tested whether HA-RAX could enhance SV40 origin-dependent plasmid DNA replication. To distinguish a newly replicated pGL3-luciferase plasmid in mammalian cells from the plasmid that was transfected into the cells, DpnI endonuclease was used because it would only cleave the transfected plasmid containing DpnI sites that were methylated during plasmid preparation in growing bacteria (26). Southern blot analysis using a radiolabeled luciferase cDNA probe revealed that pGL3-plasmid DNA replication occurred following transfection into mammalian cells and was significantly enhanced by HA-RAX (Fig. 5A). These findings indicated that RAX can stimulate SV40 promoter (origin)/enhancer-dependent DNA replication. This novel property of RAX was verified when it was shown that HA-RAX could also increase the replication of an unrelated FLAG-eIF2{alpha}/pcDNA3 plasmid in COS-7 cells (Fig. 5B). The pcDNA3 vector contains both CMV promoter/enhancer elements (for expressing the gene of interest) and the SV40 promoter (origin)/enhancer elements to allow replication of the FLAG-eIF2{alpha}/pcDNA3 in COS-7 cells. These findings help to explain how RAX can enhance expression of FLAG-eIF2{alpha}/pcDNA3 (Fig. 1A). Because RAX only slightly or moderately increased CMV promoter/enhancer activity (Figs. 2A and 4B) while significantly enhancing the FLAG-eIF2{alpha} expression, the mechanism may result primarily from RAX enhancement of SV40 promoter (origin)/enhancer dependent plasmid replication. Furthermore, because both pGL3-luciferase and the FLAG-eIF2{alpha}/pcDNA3 plasmids are different plasmid constructs, these results strongly suggest that RAX-mediated augmentation of DNA replication is not an artifact of a particular plasmid construct but rather is specific for the SV40 promoter (origin)/enhancer elements.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 5.
RAX-mediated enhancement of DNA replication. Cotransfection in COS-7 and CV-1 cells was performed as described in the legend to Figs. 1, 2, 3. The plasmids were isolated, linearized, and DpnI-digested and then probed with a radiolabeled luciferase cDNA insert or FLAG-eIF2{alpha} cDNA insert, as described under "Materials and Methods." Southern blot analysis of plasmid replication of pGL3-SV40 luciferase vector in COS-7 cells (A), FLAG-eIF2{alpha}/pcDNA3 in COS-7 cells (B), and pGL3-SV40 luciferase vector in CV-1 cells (C). Lane 1, control, the DpnI-digested pGL3-SV40 vector or FLAG-eIF2{alpha}/pcDNA3 plasmid that was isolated from bacteria preparation and used as reference for the transfected plasmid.

 

To test whether the T-Ag was required for RAX-mediated enhancement of DNA replication, plasmid replication studies were carried out in CV-1 cells that lack T-Ag expression. Results indicated that expression of the T-Ag is required for RAX-mediated enhancement of SV40 promoter (origin)/enhancer dependent DNA replication (Fig. 5C).

Northern analysis confirmed that HA-RAX synergistically increased SV40 promoter/enhancer-dependent luciferase mRNA expression in COS-7 and CV-1 cells (Fig. 6, A and B). Results in CV-1 cells indicated that although the basal level of luciferase mRNA expression was undetectable, probably because of the small amount of luciferase vector used in the cotransfection, HA-RAX synergized with the co-expressed T-Ag to stimulate mRNA expression (Fig. 6B, comparing lanes 1 and 2 with lanes 3 and 4). Because (luciferase) gene transcription primarily occurs downstream of plasmid replication, HA-RAX-enhancement of mRNA expression resulted, at least in part, from enhanced plasmid replication. As shown in Figs. 5 and 6, the magnitude of HA-RAX-mediated enhancement of mRNA expression appeared to be greater than that of plasmid DNA synthesis, especially in CV-1 cells, indicating that RAX likely stimulated SV40 regulatory element-dependent DNA transcription as well. Because the T-Ag can function in both SV40 DNA synthesis and transcriptional activation that are tightly coupled processes, RAX may cooperatively stimulate these two processes.



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 6.
RAX-mediated enhancement of mRNA expression. Cotransfection in COS-7 and CV-1 cells was performed as described in the legends to Figs. 2 and 3. Total RNAs were isolated and probed with the radiolabeled luciferase cDNA insert as described under "Materials and Methods." Northern blot analysis in COS-7 (A) and CV-1 cells (B). Luc, luciferase mRNA. Ethidium bromide staining of 18 S rRNA was used as a loading control (bottom panels).

 

DsRNA-binding Domains Are Required to Enhance Luciferase Gene Expression—RAX/PACT contains three putative dsRNA-binding motifs that are required for PKR activation (15, 16, 29). The first two dsRNA-binding motifs (dsRBM) display high homology with the first dsRBM of PKR (15, 16, 30). Lys-84 and Lys-177 are conserved lysine residues located within the first and second dsRBMs that are necessary for the dsRNA binding of RAX (15, 16, 29). Constructs containing site-directed mutation (i.e. K84A and K177A) or deletion of one or both of these domains (i.e. {Delta}II and {Delta}I/II) were created to examine a role for the dsRNA-binding property of RAX (Fig. 7A). Binding to poly(I)·poly(C)-agarose beads indicated that the first two dsRBMs were essential for dsRNA binding, whereas the third dsRBM alone failed to bind (Fig. 7B). Because only one of the first two dsRBMs of RAX is sufficient for poly(I)·poly(C) binding in vitro under physiological salt concentrations (150 mM NaCl), a potential difference in the binding affinity between wild-type RAX and mutants in only one of the dsRNA-binding domains could not be detected (Fig. 7B). However, when a more stringent binding condition (500 mM NaCl) is used, the RAX mutants display lower poly(I)·poly(C) binding than wild-type RAX in vitro (data not shown). However, whether differential dsRNA binding of the wild-type and mutant RAX proteins occurs in vivo is not yet clear. Now, using these RAX constructs, we tested whether the dsRNA-binding property can affect RAX-augmented SV40 T-Ag-dependent gene expression. pGL3-SV40 luciferase assays were performed in COS-7 cells co-transfected with the wild-type or a mutant FLAG-tagged RAX as described above. Results reveal that deletion of the first two dsRBMs completely abolishes RAX-mediated augmentation of luciferase gene expression, whereas mutating or deleting only a single such domain demonstrates reduced stimulation of expression (Fig. 7C). These findings indicate that the first two dsRNA-binding domains of RAX may be necessary for RAX-mediated enhancement of gene expression.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 7.
Mutational analysis of the role of the dsRNA-binding domains in RAX enhancement of gene expression. A, schematic representation of wild-type (wt) and mutant RAX. B, poly(I)·poly(C)-agarose bead binding assays. Expression plasmids of FLAG-tagged wild-type (Wt) and mutant RAX in the pcDEF3 vector were transfected into COS-7 cells, respectively. After 48 h cells were lysed and aliquots (50 µg) of lysates were incubated with poly(I)·poly(C)-agarose beads that were then washed with lysis buffer followed by SDS-PAGE and immunoblotting using an anti-FLAG antibody, as described under "Materials and Methods." Poly(C)-agarose bead binding of wt RAX was used as a negative control. I, input; B, poly(I)·poly(C) or poly(C)-beads bound protein. pIC, poly(I)·poly(C)-beads-binding; pC, poly(C)-bead binding. C, luciferase assays. The plasmids of FLAG-tagged wild-type and mutant RAX were co-transfected with the pGL3-SV40 luciferase reporter vector into COS-7 cells followed by luciferase assays as described in the legend to Fig. 2.

 

PKR Is Not Required for RAX-mediated Enhancement of Gene Expression—Because RAX is a cellular activator of PKR, we tested whether PKR was involved in RAX enhancement of SV40 T-Ag-dependent gene expression. A potential effect of RAX on eIF2{alpha} phosphorylation and protein synthesis was examined. COS-7 cells were transfected with either an HA-tagged RAX plasmid or an empty vector and metabolically labeled with [35S]methionine to measure steady-state levels of protein synthesis. Western blot analysis was performed using a phospho-eIF2{alpha}-specific antibody to assess eIF2{alpha} phosphorylation because eIF2{alpha} is the physiologic substrate of activated PKR (31). Results clearly demonstrate that neither global cellular protein synthesis nor PKR activity was affected by the HA-RAX expressed in COS-7 cells (Fig. 8, A and B). In addition, expression levels of the T-Ag and eIF2{alpha} in these cells were also not affected by HA-RAX expression (Fig. 8B).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 8.
RAX-mediated enhancement is PKR-independent. A, 35S-metabolic labeling. The HA-RAX/pGC1 plasmid (empty vector as a control) was co-transfected with the pGL3-SV40 luciferase vector into COS-7 cells. After 48 h, proteins in COS-7 cells that transiently express HA-tagged RAX were pulse-labeled with [35S]methionine for 60 min to assess protein synthesis. The cells were lysed followed by SDS-PAGE, Western transfer, and autoradiography. B, Western blot analysis. The same blot was also immunoblotted using antibodies against phospho-eIF2{alpha}, eIF2{alpha}, HA, and SV40 T-Ag. C, the PKR inhibitor 2-AP treatment. The co-transfection was performed as described in the legend to Fig. 2 in the presence or absence of 10 mM 2-AP followed by luciferase assays.

 

In addition to regulating protein synthesis, PKR has been reported to participate in other fundamental cell processes including signal transduction and activation of transcription factors (22). To assess whether PKR activity might play a role in RAX-mediated enhancement of reporter gene expression, 2-AP, a potent inhibitor of PKR, was tested (25). HA-RAX was co-transfected with the pGL3-SV40 luciferase reporter vector in the presence or absence of a high concentration of 2-AP (10 mM) that could inhibit PKR activity (25). Results indicated that 2-AP addition had essentially no effect on HA-RAX enhancement of luciferase gene expression (Fig. 8C). Therefore, the enhancement of reporter gene expression likely resulted from augmentation of T-Ag-dependent DNA replication and transcription rather than affecting the rate of protein synthesis. These data also indicated that RAX enhancement of DNA replication and gene expression is independent of PKR activity.

RAX Augments T-Ag-dependent Gene Expression in a Mechanism That Involves the SV40 Enhancer—Whether RAX can co-operate directly with the T-Ag was tested to determine whether a stable association between HA-RAX and T-Ag could be detected. Results indicate this was not the case (data not shown). Alternatively, a potential role of the SV40 enhancer, which has no T-Ag binding site but contains binding sites for several cellular transcription factors (32–34), was tested. HA-RAX was co-transfected with a pGL3-luciferase vector containing either the SV40 promoter (origin) element alone or the SV40 promoter (origin) and enhancer elements together. Results reveal that expression of HA-RAX augments SV40 promoter (origin)/enhancer-dependent luciferase gene expression significantly more than the expression observed from the plasmid containing the SV40 promoter (origin) element alone (Fig. 9A). Furthermore, HA-RAX also stimulates the SV40 enhancer transcriptional activity independent of the SV40 origin of replication (Fig. 9B). These data suggest that the SV40 enhancer may play a critical role in RAX-stimulated SV40 regulatory element-dependent DNA replication and transcription.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 9.
Role of the SV40 enhancer in RAX-mediated enhancement of gene expression. A, 0.1 µg of pGL3-luciferase reporter vectors containing the SV40 promoter (origin) element only (pGL3-SV40-P) and both SV40 promoter (origin) and enhancer elements (pGL3-SV40-P&E) were co-transfected with HA-RAX into COS-7 cells followed by luciferase assays as described in the legend to Fig. 2A. Luciferase expression is displayed relative to that of the vector co-transfected with pGL3-SV40-P. Western blot analysis of protein expression using antibodies against HA and SV40 T-Ag. V, empty vector pGC1; RAX, HA-RAX/pGC1. B, pSV40 E-TAL luciferase reporter vector containing the SV40 enhancer alone (pSV40-E-TAL) and pTAL control reporter vector (pTAL) were co-transfected with HA-RAX into COS-7 cells followed by luciferase assays and Western blot analysis as described in A. Luciferase expression is displayed relative to that of the vector co-transfected with pTAL vector.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results reported here support a novel, unexpected function for RAX, the cellular activator of PKR, to synergize with the T-Ag to stimulate SV40 promoter (origin)/enhancer-dependent luciferase reporter gene expression (Figs. 2, 3, 4). RAX-mediated augmentation of luciferase gene expression occurs at the level of both plasmid DNA replication (Fig. 5) and mRNA expression (Fig. 6) but independent of PKR (Fig. 8). Furthermore, using different promoter/enhancer elements (SV40, CMV, and RSV) or reporter genes (Firefly luciferase, Renilla luciferase, and FLAG-eIF2{alpha}), results confirm that RAX-mediated augmentation is specific for the SV40 promoter (origin)/enhancer elements. Therefore, in addition to its regulatory role in activating PKR, we can conclude that RAX can also function as a positive regulator of SV40 regulatory element-dependent DNA replication and gene expression.

Results were obtained in several mammalian cell lines (COS-7, CV-1, and HeLa) indicating that the T-Ag is required for RAX-mediated stimulation of SV40 promoter (origin)/enhancer-dependent DNA replication and gene expression (Figs. 2, 3, 4, 5, 6). Although a significant synergy can be observed between the RAX and T-Ag, attempts to detect a stable direct association between these two proteins was not successful. These data suggest that RAX may indirectly co-operate with the T-Ag to stimulate DNA replication and gene expression. Interestingly, ectopic expression of RAX in COS7 cells enhances the association between the T-Ag and an as yet undetermined ~75-kDa cellular protein (data not shown). Efforts are ongoing in the lab to identify this ~75-kDa protein.

It is now well recognized that a plasmid containing the SV40 promoter (origin)/enhancer elements can be assembled into the chromatin structure following the transfection of CV-1 or COS cells (35, 36). Such nucleosomal assembly indicates that plasmid DNA replication and transcription closely resembles SV40 DNA replication and transcription in vivo. Thus, RAX-mediated enhancement of gene expression may result from its stimulatory role as a cellular regulator of SV40 DNA replication and transcription in vivo. The SV40 promoter (origin)/enhancer sequence contained in the reporter vectors employed consists of an intact viral origin of DNA replication and auxiliary elements including the 21-bp repeats and the 72-bp enhancer elements (23). The T-Ag directly interacts with the viral origin region and weakly binds to the 21-bp repeats, whereas the 72-bp enhancer elements do not contain a T-Ag binding site but contain other cellular transcription factor-binding sites (e.g. a binding site for AP-1) (32, 34). Whereas the auxiliary transcriptional elements (i.e. the 21-bp repeats and the SV40 enhancer) are not essential for viral DNA replication, they can enhance replication activity and are necessary for the efficient SV40 DNA replication in vivo (33, 37–39). Interestingly, the SV40 enhancer was found to play a critical role in RAX-mediated enhancement of gene expression (Fig. 9). Thus, RAX may enhance the T-Ag function indirectly to stimulate DNA replication and transcription by altering the interaction of cellular factors (such as AP-1) with the SV40 enhancer. Recently, the SV40 enhancer (including the AP-1 element) was reported to direct chromatin remodeling in SV40 chromosomes during activation of early transcription (40). Because binding of transcriptional factor-mediated chromatin remodeling can play a role not only in transcriptional activation but also viral DNA replication, RAX may regulate (or cooperate with) cellular factors such as AP-1 that bind to the SV40 enhancer. While speculative, this could modify the nucleosomal structure and stimulate SV40 DNA replication and transcription. Further studies will be required to elucidate this mechanism.

Recently, an ~70-amino acid dsRBM that specifically binds dsRNA has been identified in a growing family of dsRNA-binding proteins, including those expressed in viruses, bacteria, and lower and higher eukaryotes (41, 42). RAX/PACT contains three such dsRBMs that possess differential dsRNA-binding properties (Fig. 7B)(29). The first two dsRBMs of RAX/PACT are essential for dsRNA binding. It is known that dsRNA-binding domains are not only necessary for dsRNA binding but also can mediate protein-protein interactions in either an RNA-dependent or an RNA-independent manner (15, 16, 29, 43, 44). These domains have been found to be critical for the function and regulation of dsRNA-binding proteins (42). For example, the dsRNA-binding domain of PKR plays an essential role in its dimerization and enzymatic activity, interactions with regulators of PKR, and targeting to the ribosome (43, 44). Whereas our results indicate that the dsRNA-binding domains of RAX are essential for its enhancement of SV40 promoter (origin)/enhancer-dependent DNA replication and gene expression (Fig. 7C), the mechanism is not yet clear.

RAX/PACT was initially discovered as a dsRNA-binding protein that interacts with and activates PKR during stress signaling (15, 16). Given this, it is surprising that PKR activity is apparently not involved in RAX-mediated enhancement of T-Ag-dependent DNA replication and gene expression (Fig. 8). This PKR-independent, "rogue" property of RAX may occur by T-Ag co-opting of RAX in the absence of an applied stress that would otherwise mediate RAX activation of PKR. Alternatively, T-Ag may somehow prevent PKR activation by potentially inactivating the PKR pathway. This latter possibility seems likely because it has been recently shown that expression of the SV40 T-Ag is able to inhibit a PKR-mediated translational block in virus-infected cells at a step downstream of eIF2{alpha} phosphorylation (45). Interestingly, dsRNA-binding proteins are reported to effect various biological functions (42). For example, the function of PKR is not limited to regulating protein synthesis but is also involved in regulating cell proliferation, signal transduction, and other cellular processes (22, 46). Other dsRNA-binding proteins are also reported to be involved in diverse biological processes including rRNA processing (e.g. RNase III), pre-mRNA editing (e.g. ADAR1), mRNA localization (e.g. Staufen), and transcriptional activation (e.g. RHA) (42, 47–50). Thus, a novel function for RAX in regulating SV40 DNA replication and transcription is not inconsistent with the "multifunctional" nature of dsRNA-binding proteins.

In summary, we have discovered a novel regulatory function for RAX, the cellular activator of PKR, in synergizing with the T-Ag to stimulate SV40 promoter (origin)/enhancer-dependent DNA replication and gene expression. RAX requires its dsRNA-binding domains and the SV40 enhancer to positively regulate SV40 DNA replication and transcription. Thus, RAX may play a PKR-independent role in regulating mammalian DNA replication and transcription because of the many similarities between the viral and cellular processes.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL54083. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Gifu Social Insurance Hospital, 1221-5 Dota, Kani, Gifu 509-0206, Japan. Back

§ To whom correspondence should be addressed: University of Florida Shands Cancer Center, P.O. Box 100232, 1600 S.W. Archer Rd., Gainesville, FL 32610-0232. Tel.: 352-846-1145; Fax: 352-846-1193; E-mail: smay{at}ufscc.ufl.edu.

1 The abbreviations used are: T-Ag, SV40 large T antigen; 2-AP, 2-aminopurine; eIF2{alpha}, the {alpha} subunit of eukaryotic initiation factor-2; HA, hemagglutinin; PKR, double-stranded RNA-dependent protein kinase; RAX, double-stranded RNA-dependent protein kinase-associated protein X; ds, double stranded; CMV, cytomegalovirus; RSV, Rous sarcoma virus; RBM, RNA-binding motif. Back


   ACKNOWLEDGMENTS
 
We thank Amanda Shanks for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fanning, E., and Knippers, R. (1992) Annu. Rev. Biochem. 61, 55–85[CrossRef][Medline] [Order article via Infotrieve]
  2. Herendeen, D., and Kelly, T. J. (1996) in Eukaryotic DNA Replication (Blow, J. J., ed) pp. 29–65, IRL Press, Oxford
  3. Simmons, D. T. (2000) Adv. Virus Res. 55, 75–134[CrossRef][Medline] [Order article via Infotrieve]
  4. Damnia, B., Mital, R., and Alwine, J. C. (1998) Mol. Cell Biol. 18, 1331–1338[Abstract/Free Full Text]
  5. Deb, S., DeLucia, A. L., Baur, C. P., Koff, A., and Tegtmeyer, P. (1986) Mol. Cell. Biol. 6, 1663–1670[Abstract/Free Full Text]
  6. DePamphilis, M. L., and Bradley, M. K. (1986) in The Papovaviridae (Salzman, N. P., ed) Vol. 1, pp. 99–246, Plenum Publishing Corp., New York
  7. Alexiadis, V. Varga-Weisz, P. D., Bonte, E., Becker, P. B., and Gruss, C. (1998) EMBO J. 17, 3428–3438[CrossRef][Medline] [Order article via Infotrieve]
  8. Hovanessian, A. G. (1989) J. Interferon Res. 9, 641–647[Medline] [Order article via Infotrieve]
  9. Clemens, M. J., and Elia, A. (1997) J. Interferon Cytokine Res. 17, 503–524[Medline] [Order article via Infotrieve]
  10. Meurs, E., Chong, K., Galabru, J., Thomas, N. S., Kerr, I. M., Williams, B. R., and Hovanessian, A. G. (1990) Cell 62, 379–390[CrossRef][Medline] [Order article via Infotrieve]
  11. Ito, T., Jagus, R., and May, W. S. (1994) Proc. Natl. Acad. Sci. U. S. A., 91 7455–7459[Abstract/Free Full Text]
  12. Hershey, J. W. B. (1991) Annu. Rev. Biochem. 60, 717–755[CrossRef][Medline] [Order article via Infotrieve]
  13. Yeung, M. C., Liu, J., and Lau, A. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12451–12455[Abstract/Free Full Text]
  14. Der, S. D., Yang, Y. L., Weissamn, C., and Williams, B. R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3279–3283[Abstract/Free Full Text]
  15. Ito, T., Yang, M., and May, W. S. (1999) J. Biol. Chem. 274, 15427–15432[Abstract/Free Full Text]
  16. Patel, R. C., and Sen, G. C. (1998) EMBO J. 17, 4379–4390[CrossRef][Medline] [Order article via Infotrieve]
  17. Patel, C. V., Handy, I., Goldsmith, T., and Patel, R. C. (2000) J. Biol. Chem. 275, 37993–37998[Abstract/Free Full Text]
  18. Ruvolo, P. P., Gao, F., Blalock, W. L., Deng, X., and May, W. S. (2001) J. Biol. Chem. 276, 11754–11758[Abstract/Free Full Text]
  19. Judware, R., and Petryshyn, R. (1991) Mol. Cell. Biol. 11, 3259–3267[Abstract/Free Full Text]
  20. Koromilas, A. E., Roy, S., Barber, G. N., Katze, M. G., and Sonenberg, N. (1992) Science 257, 1685–1689[Abstract/Free Full Text]
  21. Jagus, R., Joshi, B., and Barber, G. N. (1999) Int. J. Biochem. Cell Biol. 31 123–138[CrossRef][Medline] [Order article via Infotrieve]
  22. Williams, B. R. (2001) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;2001/89/re2
  23. Pauly, M., Treger, M., Westhof, E., and Chambon, P. (1992) Nucleic Acids Res. 20, 975–982[Abstract/Free Full Text]
  24. Goldman, L. A., Cutrone, E. C., Kotenko, S. V., Krause, C. D., and Langer, J. A. (1996) BioTechniques 21, 1013–1015[Medline] [Order article via Infotrieve]
  25. Hu, Y., and Conway, T. W. (1993) J. Interferon Res. 13, 323–328[Medline] [Order article via Infotrieve]
  26. Li, J. J., and Kelly, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 6973–6977[Abstract/Free Full Text]
  27. Thomis, D. C., and Samuel, C. E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10837–10841[Abstract/Free Full Text]
  28. Gluzman, Y. (1981) Cell 23, 175–182[CrossRef][Medline] [Order article via Infotrieve]
  29. Peters, G. A., Hartmann, R., Qin, J., and Sen, G. C. (2001) Mol. Cell. Biol. 21, 1908–1920[Abstract/Free Full Text]
  30. Green, S. R., and Mathews, M. B. (1992) Genes Dev. 6, 2478–2490[Abstract/Free Full Text]
  31. Williams, D. D., Pavitt, G. D., and Proud, C. G. (2001) J. Biol. Chem. 276, 3733–3742[Abstract/Free Full Text]
  32. Herr, W., and Clarke, J. (1986) Cell 45, 461–470[CrossRef][Medline] [Order article via Infotrieve]
  33. Haas, M. W., Ramanujam, P., Chandrasekharappa, S. C., and Subramanian, K. N. (1991) Virology 180, 41–48[CrossRef][Medline] [Order article via Infotrieve]
  34. DePamphilis, M. L. (1993) Annu. Rev. Biochem. 62, 29–63[CrossRef][Medline] [Order article via Infotrieve]
  35. Cereghini, S., and Yaniv, M. (1984) EMBO J. 3, 1243–1253[Medline] [Order article via Infotrieve]
  36. Gruss, C., Gutierrez, C., Burhans, W. C., DePamphilis, M. L., Koller, T., and Sogo, J. M. (1990) EMBO J. 9, 2911–2922[Medline] [Order article via Infotrieve]
  37. Cheng, L., and Kelly, T. J. (1989) Cell 59, 541–551[CrossRef][Medline] [Order article via Infotrieve]
  38. Guo, Z. S., and DePamphilis, M. L. (1992) Mol. Cell Biol. 12, 2514–2524[Abstract/Free Full Text]
  39. Turner, W. J., and Woodworth, M. E. (2001) J. Virol. 75, 5638–5645[Abstract/Free Full Text]
  40. Milavetz, B. I. (2002) Virology 294, 170–179[CrossRef][Medline] [Order article via Infotrieve]
  41. St. Johnston, D., Brown, N. H., Gall, J. G., and Jantsch, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10979–10983[Abstract/Free Full Text]
  42. Fierro-Monti, I., and Mathews, M. B. (2000) Trends Biol. Sci. 25, 241–246
  43. Tian, B., and Mathews, M. B. (2001) J. Biol. Chem. 276, 9936–9944[Abstract/Free Full Text]
  44. Ung, T. L., Cao, C., Lu, J. Ozato, K., and Dever, T. E. (2001) EMBO J. 20, 3728–3737[CrossRef][Medline] [Order article via Infotrieve]
  45. Swaminathan, S., Rajan, P., Savinova, O., Jagus, R., and Thimmapaya, B. (1996) Virology 219, 321–323[CrossRef][Medline] [Order article via Infotrieve]
  46. Proud, C. G. (1995) Trends Biochem. Sci. 20, 241–246[CrossRef][Medline] [Order article via Infotrieve]
  47. Robertson, H. D. (1982) Cell 30, 669–672[CrossRef][Medline] [Order article via Infotrieve]
  48. Bass, B. L., and Weintraub, H. (1988) Cell 55, 1089–1098[CrossRef][Medline] [Order article via Infotrieve]
  49. St. Johnston, D., Beuchle, D., and Nusslein-Volhard, C. (1991) Cell 66, 51–63[CrossRef][Medline] [Order article via Infotrieve]
  50. Nakajima, T., Uchida, C., Anderson, S. F., Lee, C. G., Hurwitz, J., Parvin, J. D., and Montminy, M. (1997) Cell 90, 1107–1112[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
R. L. Bennett, W. L. Blalock, and W. S. May
Serine 18 Phosphorylation of RAX, the PKR Activator, Is Required for PKR Activation and Consequent Translation Inhibition
J. Biol. Chem., October 8, 2004; 279(41): 42687 - 42693.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/40/38325    most recent
M303420200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, M.
Right arrow Articles by May, W. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, M.
Right arrow Articles by May, W. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement