From the Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
Received for publication, November 20, 2002 , and in revised form, May 12, 2003.
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ABSTRACT |
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INTRODUCTION |
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The protein region responsible for the transcriptional activation (called the "transactivation domain" (TAD)) of p65 has been mapped in their unique C-terminal region containing at least two TADs within its C-terminal 120 amino acids, termed TA1 and TA2 (57). It has been revealed that the p65 TAD interacts with transcriptional coactivators, such as p300/CREB-binding protein (8, 9) and FUS/TLS (10), and general transcription factors, including TBP (11) and TFIIB (7, 12). Interaction of p65 with these factors stimulates transcription by initiating chromatin remodeling or by recruiting RNA polymerase II. In an unexpected scenario, the involvement of ubiquitination has recently been implicated in the regulation of TADs of some transcriptional activators such as VP16 (13, 14), Myc (13), and nuclear receptors (1517), either directly or indirectly (for an excellent review, see Conaway et al. (18)).
In this context, we became interested in AO7, which we have identified as one of the interacting proteins with the p65 TAD in the CytoTrapTM yeast two-hybrid screen. We have adopted this alternative screening method instead of the commonly used method utilizing the Gal4 transcription system, because the p65 TAD is functional in the yeast (10, 19). AO7 encodes a protein containing a RING finger domain and is ubiquitously expressed in various tissues (20). AO7 was initially identified in the yeast two-hybrid screen of a murine T cell library by using UbcH5b, an E2 enzyme, as bait (20). Although the target protein for the uniquitination complex involving AO7 and UbcH5b still remains to be determined, AO7 has been shown to act as a putative E3 ligase at least in vitro (20). We found that AO7 acts as a mediator of p65 transactivation. A possible mechanism of its action is discussed.
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EXPERIMENTAL PROCEDURES |
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Yeast Two-hybrid ScreeningThe CytoTrapTM (Stratagene) yeast screening was performed with human lung cDNA library (Stratagene) and pSos-(262551) as a bait according to the manufacturer's instructions and the method previously described (24). Saccharomyces cerevisiae strain cdc25H was transformed sequentially with pSos-p65-(262551) and human lung cDNA library fused to the pMyr plasmid containing the myristylation sequence of v-Src. Since the cdc25H yeast cell contains a temperature-sensitive mutant of the yeast homologue (cdc25H) of human hSos, it cannot grow at 37 °C. Positive clones were selected by the growth ability of cdc25H cells on galactose plate at 37 °C, in which the hSos-p65-(262551) protein is recruited to the plasma membrane because of the interaction with the myristoylated protein encoded by a pMyr clone selected from the target library, thereby complementing the cdc25 defect and allowing the growth of the cdc25H yeast clone at 37 °C due to activation of the Ras-signaling pathway. The pMyr plasmids were rescued from positive colonies and identified by nucleotide sequencing.
Isolation of Full-length Human AO7Human full-length AO7 cDNA was obtained by PCR using oligonucleotide primers, 5'-atggcggcgtctgcgtctgcagc-3' (forward) and 5'-aaggccaaatatctttattgcctccc-3' (reverse), and a full-length human cDNA library prepared from neuroblastoma cell line SK-N-MC (25) (a generous gift from S. Sugano, University of Tokyo) as a template. The nucleotide sequences of oligonucleotide primers for full-length AO7 cDNA were designed based on the identity of the prey plasmid with human AO7 homologue, RNF25 (ring finger protein 25). PCR was performed using Expand High FidelityTM system (Roche Applied Science), and the PCR products were cloned into pGEM vector (Promega). Nucleotide sequencing was performed by the ABI PRISM dye terminator cycle sequencing ready reaction kit with an Applied Biosystems 313 automated DNA sequencer (ABI). Nucleotide sequence of human AO7 was determined using forward and reverse M13 primers and five internal sequencing primers: 5'-ggtgtgcagtgtccagtgtg-3' (forward 1), 5'-caaagggaggggagtgccacg-3' (forward 2), 5-tgttatctgtgagaatttcc-3' (reverse 1), 5'-gcgcaagctctctgcactgg-3' (reverse 2), and 5'-cgtggcactcccctccctttg-3' (reverse 3), corresponding to the various portions of human AO7 cDNA.
Cell Culture and Transfection293 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum,
100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were transfected
with various plasmids using Fugene-6TM transfection reagent (Roche
Molecular Biochemicals) according to the manufacturer's instructions. At 24 h
post-transfection, the cells were harvested, and the whole cell extracts were
prepared for the luciferase assay. The luciferase activity was measured by the
luciferase assay system (Promega) as previously described
(10,
19). The luciferase enzyme
activity was normalized based on the protein concentration of whole cell
extracts determined by using the Protein Assay kit (Bio-Rad). The data are
presented as the -fold increase in luciferase activities (means ± S.D.)
relative to control of three independent transfections. Recombinant human
IL-1 and TNF were purchased from Roche Applied Science.
ImmunofluorescenceCells were immunostained with rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (primary antibody) and fluorescein isothiocyanate-labeled goat anti-rabbit IgG (Cappel) (secondary antibody) to demonstrate the localization of p65 as described previously (26). In order to examine the subcellular localization of AO7, pEGFP-AO7 was transfected into 293 cells, and the intracellular localization of green fluorescence protein (GFP) (excitation at 490 nm and emission at 520 nm) was examined.
In Vitro Binding AssaypGEX-AO7, its derivatives, and
pGEX-p65- (441521) were transformed in Escherichia coli strain
DH5 following induction with 0.1 mM
isopropyl-1-thio--D-galactopyranoside at 25 °C for 6 h.
Recombinant GST proteins were purified by affinity chromatography on
glutathione-agarose beads as described previously
(27). In vitro
protein-protein interaction assays were performed as reported
(27). Briefly, the
[35S]methionine-labeled p65 and AO7 proteins were incubated with
immobilized GST fusion proteins overnight at 4 °C in 1 ml of modified
HEMNK buffer (40 mM HEPES-KOH (pH 7.5), 50 mM KCl, 5
mM MgCl2, 0.2 mM EDTA, 0.1% Nonidet P-40, 1
mM dithiothreitol, 1x CompleteTM protease inhibitors
(Roche Applied Science)). After five washes with 1 ml of HEMNK buffer, bound
radiolabeled proteins were eluted with 20 µl of Laemmli sample buffer,
boiled for 3 min, and resolved by 10% SDS-PAGE.
Coimmunoprecipitation and Western Blot AssaysIn order to examine the protein-protein interaction in cultured 293 cells, pcDNA-AO7-(1459) was transfected, and cells were cultured for 48 h with or without 20 ng/ml of TNF stimulation for 30 min before the harvest. The cell extract was prepared by treatment with the lysis buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, and 1x CompleteTM protease inhibitors) containing 120 mM NaCl (low salt) or 300 mM (high salt). The cell lysate was incubated in the same buffer with 20 µl of anti-FLAG M2 Affinity Gel (Sigma) at 4 °C for 1 h with continuous rotation at 10 rpm. The beads were washed three times with 1 ml of lysis buffer. The antibody-bound complex was eluted by boiling in 1x Laemmli sample buffer. The immunoprecipitated proteins were resolved by 8% SDS-PAGE and transferred on nitrocellulose membrane (Hybond-C; Amersham Biosciences). The membrane was incubated with rabbit polyclonal anti-p65 (C-terminal) antibody (Santa Cruz Biotechnology), and immunoreactive proteins were visualized by enhanced chemiluminescence (SuperSignal; Pierce) as described previously (28). To detect the level of endogenous p65 and FLAG-tagged AO7 expression, rabbit polyclonal anti-p65 antibody (Santa Cruz) and mouse monoclonal anti-FLAG (Sigma) were used.
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RESULTS |
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We screened 3.3 million transformants from the human lung cDNA
library. Nucleotide sequence determination and comparison with GenBankTM
databases (National Center for Biotechnology Information) revealed human
I
B
(seven clones) and only one clone encoding human homologue of
mouse AO7 (the corresponding protein of AO7 in human and mouse has also been
termed RNF25; GenBankTM accession number NM022453). We isolated a
full-length human AO7 cDNA clone from the library derived from human
neuroblastoma cell line SK-N-MC
(25) for further study.
To confirm the interaction between p65 and AO7 in vitro, we
performed the GST pull-down assay. GST fusion proteins with various portions
of AO7 (Fig. 1A) were
synthesized in E. coli, immobilized on glutathione-Sepharose beads
and incubated with the [35S]methionine-labeled p65 (full-length).
Fig. 1B shows that p65
bound to GST-AO7-(1459), GST-AO7-(212459), and
GST-AO7-(341459), although the binding affinities of AO7 and some
mutants with p65 appeared to be lower than that of IB
. However,
p65 did not significantly bind to GST-AO7-(1219) containing the RING
finger domain. No p65 binding was detected with GST alone. To further confirm
this interaction, we also performed the reverse GST pull-down assay using
GST-p65-(441521) and the [35S]methionine-labeled
AO7-(1459), AO7-(1219), and AO7-(212459). In
Fig. 1C,
GST-p65-(441521) bound to AO7-(1459) and AO7-(212459) but
not to AO7-(1219). These results suggested that the minimal region of
AO7 responsible for the interaction with the C-terminal region of p65 resides
within the C-terminal amino acid sequence 341459, overlapping with the
Pro-rich region.
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AO7 Interacts with p65 in VivoTo examine whether AO7
interacts with p65 in vivo, 293 cells were transfected with
pcDNA-AO7-(1459), expressing the FLAG-tagged full-length AO7 and
harvested for the co-immunoprecipitation assay. As demonstrated in
Fig. 2, AO7-(1459) was
co-immunoprecipitated with p65, and this interaction was not significantly
decreased even at the higher salt concentration (lane 4), suggesting
the strong affinity between p65 and AO7. When stimulated with TNF, which
causes the nuclear translocation of NF-B and phosphorylation of p65
(2931) a greater interaction between AO7 and p65 was observed (compare
lanes 2 and 6), suggesting that AO7 interacts with p65 in
the nucleus. Interestingly, this TNF-induced augmentation of the interaction
between p65 and AO7 was significantly reduced at higher salt concentration
(compare lanes 6 and 8), implying that the increased
affinity between p65 and AO7 may be due to the increased electrostatic
interaction such as by phosphorylation of p65.
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AO7 Augments the NF-B-dependent Gene Expression Induced
by NIKSince mouse AO7 was initially identified as an interacting
protein with ubiquitin-conjugating enzyme (E2), UbcH5B, and involved in
E2-dependent ubiquitination through its RING finger domain, we first asked if
AO7 is involved in the NF-
B-induced transcription. We performed a
transient luciferase assay using pGL34
B-luc reporter plasmid, in
which luciferase reporter gene is under the control of NF-
B.
Fig. 3 shows that
cotransfection with pcDNA-AO7-(1459) activated NF-
B-dependent
gene expression upon NIK overexpression in a dose-dependent manner for the
amount of AO7-expressing plasmid. Western blot analysis of the transfected
cell lysate revealed no increase in the protein level of endogenous p65
(Fig. 3A). The similar
effect of AO7 was observed when NF-
B was stimulated with IL-1
or
TNF (Fig. 3B). To
further investigate the AO7 action, we created AO7 mutant constructs, and
their effects on the NIK-induced NF-
B activation were examined. As
shown in Fig. 3C,
neither the C-terminal nor N-terminal truncated AO7 mutant enhanced
NF-
B activation mediated by NIK or exhibited dominant negative effect.
These findings indicate that both the C-terminal and the N-terminal regions of
AO7 are required for its effect on the NF-
B activity.
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Effect of AO7 Is Mediated by the p65 Subunit of
NF-BTo investigate whether AO7 enhances the action
of NF-
B through acting on NIK-mediated signaling pathway or the
transcriptional activity of p65, we examined the effect of AO7 on
NF-
B-dependent gene expression when p65 is overexpressed. As shown in
Fig. 4A, AO7 augmented
the NF-
B-dependent gene expression when p65-expressing plasmid was
cotransfected, indicating that the effect of AO7 does not depend on the
signaling cascade involving NIK and IKK complex. Using a control reporter
plasmid, 4
Bm-luc, in which all four
B sites were mutated, no
effect of AO7 was observed even upon overexpression of p65. These results
indicate that AO7 stimulates the transcriptional activity of p65 but not
through the basal transcriptional machinery.
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AO7 Is Expressed Abundantly in the NucleusIn order to
examine the subcellular localization of AO7, the full-length AO7 in fusion
with GFP was expressed in 293 cells. As shown in
Fig. 4B, AO7 was
detected predominantly in the nucleus, although the cytoplasmic localization
was also evident. This intracellular distribution of AO7 was not altered by
TNF stimulation. In addition, AO7 was colocalized with p65 when the nuclear
translocation of NF-B was induced by the treatment with TNF. These
findings suggest that AO7 may interact with p65 in the nucleus.
AO7 Activates NF-B through the Transactivation Domain
of p65 (RelA)To further investigate the effect of AO7 on
NF-
B, various p65 mutants fused to Gal4BD
(Fig. 5A) were
cotransfected into 293 cells with the reporter plasmid, pFRluc, in which
luciferase gene expression is under the control of Gal4, with or without the
AO7 expression plasmid pcDNA-AO7- (1459). As shown in
Fig. 5B, AO7 enhanced
the Gal4-p65-dependent gene expression in a dose-dependent manner, and
truncation of either the N-terminal (containing RING finger motif and RWD
domain) or C-terminal (containing the Pro-rich region) half of AO7 abolished
its action. These findings indicate that both the N- and C-terminal regions of
AO7 are indispensable for its action. Moreover, these AO7 mutants did not
block the transcriptional activity of p65, which is consistent with the
results in Fig. 3B.
There was no significant effect of AO7 on the VP16-mediated transactivation
(data not shown).
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We then explored the region of p65 responsible for the AO7-mediated
transcriptional enhancement using various Gal4-p65 mutants. There was no
significant effect of AO7 on Gal4-p65N, containing p65-(1286) and
lacking the TA domain (Fig.
5C). In contrast, AO7 stimulated the transcriptional
activity of Gal4-p65C, containing nuclear localization signal, TA1, and TA2,
in a dose-dependent manner (Fig.
5D). Further truncation and deletion of the C-terminal
region of p65 revealed that both TA1 and TA2 are required for the action of
AO7 (Fig. 5,
EG). Notably, since the extent of augmentation by
AO7 for pGal4-p65C2 (8.3-fold) was greater than that for pGal4-p65C (12-fold)
(Fig. 5, compare D and
F), the central region, containing nuclear localization
signal and thus serving as the target of IB, is fully dispensable for
the action of AO7, indicating that AO7 is unlikely to be involved in the
I
B degradation. Moreover, since p65-(441521) is shown to be
sufficient for the interaction with AO7
(Fig. 1C), at least
in vitro, the action of AO7 may require additional protein(s)
interacting with TA1.
Effects of Mutation in the RING Finger Domain of AO7 AO7 contains a typical RING finger domain of the RING-H2 category, forming an interleaved zinc-binding site with six Cys residues and two His residues in the middle (C3H2C3) (Fig. 6A). These Cys and His residues of AO7 were shown to be crucial for its function such as ubiquitination and E2 binding (20). We thus introduced Cys substitutions and yielded two mutants: AO7(C159S), in which Cys at the irrelevant amino acid position 159 for RING was substituted by Ser, and AO7(C161S), in which Cys at 161, a crucial amino acid for the formation of RING and indispensable for ubiquitination and E2 binding, was substituted by Ser (20).
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In Fig. 6B, the effects of these AO7 mutants were examined. Whereas the Cys mutation at 159, AO7(C159S), did not significantly affect the action of AO7, the Cys mutation at 161, substituting a crucial Cys for RING-H2 formation, exhibited no such action. At the higher amounts of AO7(C161S), the gene expression mediated by Gal4-p65 was inhibited in a dose-dependent manner, suggesting the dominant negative characteristics for this mutant. These findings indicated that the RING-H2 domain of AO7 is important for the action of AO7 and suggested that ubiquitination might be necessary for the augmentation of p65 transcriptional activity by AO7.
Selective Action of AO7 on p65To further analyze the action of AO7, we examined whether AO7 could activate transcription when tethered to DNA. As shown in Fig. 7A, Gal4-AO7, containing the full-length AO7 fused to Gal4BD, did not activate transcription from a minimal promoter containing Gal4 binding sites, suggesting that AO7 does not act as a general coactivator of transcription. When pGal4-AO7 was cotransfected with pSport-p65, expressing full-length p65, the extent of gene induction was greatly augmented (up to 10-fold) in a dose-dependent manner for the amount of pSport-p65 (Fig. 7B). When it was cotransfected with pVP16-p65, expressing a p65-VP16 fusion protein, a similar extent of gene induction (6-fold) was observed. However, when pVP16 was cotransfected with pGal4-AO7, no such effect was observed, presumably because of the lack of AO7 binding to VP16. Together with the results in Fig. 5, these observations indicate that AO7 interacts with p65 in cells and selectively supports the transactivation of p65.
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DISCUSSION |
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AO7 was initially isolated as E2-interacting protein by conventional yeast
two-hybrid screen and shown to bind UbcH5b through its RING finger motif
(20). Although AO7 is known to
accept the ubiquitin modification by E2 (UbcH5b), its biological function
in vivo has not been elucidated. It was thus proposed that AO7 might
act as an E3-like factor. However, its target proteins have not been found. In
this study, we found that the RING finger motif of AO7 was not involved in the
binding to p65 but required for the p65-mediated transactivation, suggesting a
possibility that the AO7 might mediate the ubiquitination of p65 and enhance
its transcriptional activity. However, we could not detect direct
ubiquitination of p65 by AO7 or any change in the stability of p65 (data not
shown). Therefore, the mechanism by which AO7 participates in the
NF-B-mediated transactivation is currently unknown.
Accumulating evidence that ubiquitin-modification plays a role in
transcriptional regulation has been demonstrated
(1317).
Ubiquitination appears to control transcription not only by mechanisms
involving ubiquitin-dependent destruction of transcription factors or their
inhibitors by proteasome but also by an intriguing mechanism independent of
the proteasome (for a review, see Ref.
18). For example, most of the
NF-B activation pathways involve ubiquitination of its inhibitor
I
Bs followed by their degradation by proteasome and the generation of
p50 and p52, DNA-binding subunits of NF-
B, from their precursors is
mediated by ubiquitination-dependent proteolysis
(3,
4). It is possible that AO7 may
facilitate protein processing of these proteins. Another possibility by which
AO7 activates NF-
B is ubiquitination and degradation of corepressors
such as Sin3A, N-CoR, or, more selectively, Groucho family proteins
(19). In fact, Siah-2, a RING
finger containing E3-like protein, is known to promote the ubiquitination of
N-CoR and derepresses the gene expression
(32).
There are many proteins that possess E3 ligase activity and inhibit the action of transcription factors. For example, the p53 inhibitor Mdm2, a RING finger-containing protein, inhibits the transcriptional activity of p53 by promoting ubiquitination and degradation of p53 (33). Another E3 protein, RING finger LIM domain-binding protein, inhibits the transcriptional activity of LIM homeodomain transcription factor family by interacting with the LIM cofactor and promoting its ubiquitination (34). Similarly, WWP1, a HECT family E3 ligase, binds to the lung Krüppel-like factor through its inhibitory domain and inhibits its transcriptional activity, although ubiquitination has not been demonstrated (35).
Interestingly, there are a number of reports suggesting a positive correlation between the ubiquitin-modification and the transcriptional activation. For example, although direct ubiquitination has not been demonstrated, Molinari et al. (36) demonstrated that half-lives of heterologous transactivators such as Gal4-VP16, Gal4-p65, Gal4-p53, and Gal4-CTF inversely correlated with the potency of their TADs. In addition, it was demonstrated that the transactivation of VP16 TAD requires SCFMet30, a yeast E3, and fusion of a single ubiquitin moiety was sufficient to complement the deficiency of the SCFMet30 gene (14). These findings indicate that TAD-dependent ubiquitination may not only serve as a signal for its destruction but also be required for its function as an activator. Other examples include the involvement of E3 ligases, Rsp5/hPRF, E6-AP, and SNURF/RNF4, in the transactivation of various nuclear receptors (1517). In addition, since a TAFII250 mutant lacking the ubiquitination catalytic activity failed to support the transactivation of Dorsal, a Drosophila homologue of RelA (37), the ubiquitination of histone H1 by TAFII250 is considered to be essential for the action of Dorsal.
Taken together, our findings raise a possibility that AO7 may support the
transcriptional activity of NF-B by several mechanisms. One such
mechanism may be that the activated NF-
B interacts in the nucleus with
various factors including general transcription factors, coactivators, and
AO7. Formation of this complex may allow AO7 to direct ubiquitin modification
of some of these factors, thus dissociating the transcriptional activator
complex to initiate a subsequent step of transcription such as elongation. AO7
may also act as a scaffold protein for the interaction of transcription
factors involved in the initiation complex. Further investigations are
required to elucidate the mechanism by which AO7 supports the action of
NF-
B and its biochemical actions.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 81-52-853-8204; Fax:
81-52-859-1235; E-mail:
tokamoto{at}med.nagoya-cu.ac.jp.
1 The abbreviations used are: IL, interleukin; TAD, transcriptional
activation domain; NIK, NF-B-inducing kinase; TNF, tumor necrosis
factor; IKK, I
B kinase; GST, glutathione S-transferase;
Gal4BD, Gal4 DNA binding domain; GFP, green fluorescence protein; luc,
luciferase; CREB, cAMP-response element-binding protein; E2, ubiquitin carrier
protein; E3, ubiquitin-protein isopeptide ligase; hSos, human Sos.
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ACKNOWLEDGMENTS |
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REFERENCES |
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