JOURNAL OF VIROLOGY, Dec. 2007, p. 13735–13742
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 24
The Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Requires
the Ubiquitin-Conjugating Enzyme Ubc13 for NF-?B Activation?
Noula Shembade,1Nicole S. Harhaj,1Masahiro Yamamoto,2Shizuo Akira,2and Edward W. Harhaj1*
Department of Microbiology and Immunology, Sylvester Comprehensive Cancer Center, the University of Miami, Miller School of
Medicine, Miami, Florida 33136,1and Department of Host Defense, Research Institute for Microbial Diseases,
Osaka University, Osaka 565-0871, Japan2
Received 27 July 2007/Accepted 2 October 2007
Ubiquitination of the human T-cell leukemia virus 1 Tax oncoprotein provides an important regulatory
mechanism that promotes the Tax-mediated activation of NF-?B. However, the type of polyubiquitin chain
linkages and the host factors that are required for Tax ubiquitination have not been identified. Here, we
demonstrate that Tax polyubiquitin chains are composed predominantly of lysine 63-linked chains. Further-
more, the ubiquitination of Tax is critically dependent on the E2 ubiquitin-conjugating enzyme Ubc13. Tax
interacts with Ubc13, and small interfering RNA-mediated knockdown of Ubc13 expression abrogates Tax
ubiquitination and the activation of NF-?B. Mouse fibroblasts lacking Ubc13 exhibit impaired Tax activation
of NF-?B despite normal tumor necrosis factor- and interleukin-1-mediated NF-?B activation. Finally, the
interaction of Tax with NEMO is disrupted in the absence of Tax ubiquitination and Ubc13 expression,
suggesting that Tax ubiquitination is critical for NEMO binding. Collectively, our results reveal that Ubc13 is
essential for Tax ubiquitination, its interaction with NEMO, and Tax-mediated NF-?B activation.
Human T-cell leukemia virus type 1 (HTLV-1) is the etio-
logical agent of adult T-cell leukemia and a neuroinflammatory
disease termed HTLV-1-associated myelopathy/tropical spas-
tic paraparesis (16, 40, 60). HTLV-1 encodes a regulatory
protein, Tax, that plays an essential role in regulating viral gene
expression (59). Tax also functions as a potent oncogene by
interacting with and modulating the function of components of
signaling pathways and the cell cycle machinery (29, 36). One
of the key signaling pathways targeted by Tax to facilitate cell
transformation is NF-?B (21, 50). Tax mutants defective in
NF-?B activation are unable to immortalize primary T cells
(44). Furthermore, pharmacological inhibition of NF-?B in
HTLV-1-transformed cell lines and leukemic cells from adult
T-cell leukemia patients provokes an apoptotic response (37).
Therefore, NF-?B is required for the Tax-mediated transfor-
mation of T cells and the survival of HTLV-1-transformed
NF-?B is a family of transcription factors that play diverse
roles in innate and adaptive immunity. NF-?B serves as a
paradigm of how latent transcription factors in the cytoplasm
can be rapidly activated and mobilized to the nucleus to acti-
vate target genes. In the canonical NF-?B pathway, the I?B
family member, I?B?, interacts with NF-?B dimers and pre-
vents nuclear translocation and DNA binding (45). In response
to specific signals, such as inflammatory cytokines, or infection
with pathogens, I?B? is phosphorylated by a multisubunit ki-
nase complex, I?B kinase (IKK), consisting of two catalytic
subunits, IKK? and IKK?, and a regulatory subunit, NF-?B
essential modulator (NEMO) or IKK? (18, 26). The phospho-
rylation of I?B? by IKK triggers polyubiquitination and deg-
radation by the 26S proteasome, thus allowing NF-?B to enter
the nucleus. An important regulatory mechanism for NF-?B
activation may be provided by lysine 63 (K63)-linked polyubiq-
uitination of signaling proteins, such as tumor necrosis factor
(TNF)-associated factor 6 (TRAF6) (1). TRAF6 functions as
an E3 ligase and requires a heterodimeric ubiquitin-conjugat-
ing enzyme complex composed of Ubc13 and the Ubc13-re-
lated protein Uev1A (8). TRAF6 plays an essential role in
interleukin-1 (IL-1), lipopolysaccharide, and CD40-mediated
NF-?B activation (35). However, gene-targeting studies in
mice have revealed that Ubc13 is dispensable for NF-?B acti-
vation, at least in fibroblasts and B lymphocytes (12, 56).
Ubc13-deficient thymocytes displayed a modest reduction in
NF-?B activation in response to T-cell receptor stimulation
In the noncanonical pathway, a more restrictive set of signals
provided by TNF receptor family members leads to the acti-
vation of the NF-?B-inducing kinase and IKK?, resulting in
the limited degradation or processing of the p100 I?B family
member to p52 (47, 55). The noncanonical pathway plays im-
portant roles in lymphoid organogenesis and B-cell maturation
and survival (2, 7).
Tax is a potent activator of both the canonical and non-
canonical NF-?B pathways. Tax activates the IKK complex by
directly interacting with NEMO, an event that is central to the
activation of both canonical and noncanonical pathways by Tax
(23, 30). Although the exact mechanism by which Tax activates
IKK remains unclear, Tax may recruit upstream kinases, such
as transforming growth factor ?-activated kinase 1 (15, 51, 52).
In addition to providing an intracellular stimulus for IKK ac-
tivation, Tax also opposes IKK-negative regulatory mecha-
nisms, such as that exerted by the phosphatase PP2A (11).
Recent studies have also demonstrated that the Tax activation
of NF-?B is regulated by Tax mono- and polyubiquitination (6,
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Sylvester Comprehensive Cancer Center, the
University of Miami, Miller School of Medicine, 1550 NW 10 Avenue,
Miami, FL 33136. Phone: (305) 243-7893. Fax: (305) 243-6410. E-mail:
?Published ahead of print on 17 October 2007.
25, 34, 39, 41). Mutation of the lysine ubiquitin acceptor sites
within Tax renders Tax unable to activate both canonical and
noncanonical NF-?B pathways (25). Although the exact link-
ages of the Tax polyubiquitin chains have not been established,
Tax ubiquitination does not promote the destabilization of
Tax, suggesting that it plays a regulatory role.
In this paper, we have identified Tax polyubiquitin chains as
predominantly K63-linked. The E2 ubiquitin-conjugating en-
zyme Ubc13 was found to be required for the ubiquitination of
Tax. Interestingly, Tax interacted with Ubc13 in transfected
cells and HTLV-1-transformed cell lines. NF-?B activation was
disrupted by small interfering RNA (siRNA)-mediated knock-
down of Ubc13 in Tax-expressing cells and in an HTLV-1-
transformed cell line. Consistent with these results, Tax was
deficient in NF-?B activation in Ubc13 knockout fibroblasts,
despite normal TNF-? and IL-1-mediated activation of NF-?B
in these cells. Finally, in the absence of Ubc13, Tax interaction
with NEMO was abrogated, indicating that Tax ubiquitination
is essential for NEMO interaction and NF-?B activation.
MATERIALS AND METHODS
Biological reagents and antibodies. Jurkat E6-1 and 293T cells were obtained
from ATCC (Manassas, VA). The HTLV-1-transformed cell line C8166 was
described previously (25). Jurkat and C8166 cells were cultured in RPMI me-
dium (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine
serum, 100 U/ml penicillin, and 100 ?g/ml streptomycin (Invitrogen, Carlsbad,
CA). 293T cells and mouse embryonic fibroblasts (MEFs) were cultured in
Dulbecco’s modified Eagle’s medium (Mediatech, Inc.) supplemented with 10%
fetal bovine serum, 100 U/ml penicillin, and 100 ?g/ml streptomycin. The mono-
clonal anti-Tax antibody was prepared from a Tax hybridoma (168B17-46-34)
that was obtained from the AIDS Research and Reference Program, NIAID,
National Institutes of Health. The phospho-I?B? antibody (14D4) was obtained
from Cell Signaling Technology (Beverly, MA). The monoclonal p100 antibody
was purchased from Upstate/Millipore (Charlottesville, VA). Normal rabbit im-
munoglobulin G, anti-I?B? (C-21), and NEMO (FL-419) antibodies were pur-
chased from Santa Cruz (Santa Cruz, CA). The anti-Ubc13 antibody (clone
4E11) was obtained from Invitrogen/Zymed. Anti-hemagglutinin (HA) antibody
(clone 12CA5) was purchased from Roche (Indianapolis, IN). Anti-ubiquitin
antibody was purchased from Stressgen/Assay Designs (San Diego, CA). The
?-actin antibody was purchased from Abcam (Cambridge, MA). Recombinant
TNF-? and IL-1? were purchased from R&D Systems (Minneapolis, MN).
Control siRNA (siControl nontargeting siRNA no. 1) and siGenome SMART-
pool Ubc13 siRNA were purchased from Dharmacon (Lafayette, CO).
Plasmids. pCMV4-Tax, pCLXSN-Tax, pCMV4-p100, HA-ubiquitin (HA-Ub)
and HA-NEMO have all been described previously (23, 25, 53). Flag-TRAF6 was
provided by Khaled Tolba. HA-Ubc13 and HA-Ubc13 C87A were kindly pro-
vided by James Chen (8). The pSG-5 Tax wild type and Tax K10R plasmids were
gifts from Claudine Pique (6). The retroviral vector expressing Cre (pMRXP-
Cre) has been previously described (56). HA-Ub K63-only and HA-Ub K48-only
were constructed by replacing all lysines in HA-Ub with arginines, except for
lysine 48 (K48-only) and lysine 63 (K63-only), using a QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA). All mutations were confirmed by
DNA sequencing. pSuppressor Retro-GFP siRNA and pSuppressor Retro-
Ubc13 siRNA plasmids were generated by cloning siRNA sequences specific for
green fluorescent protein (GFP) and Ubc13 into the Xho and XbaI sites in
pSuppressor Retro (Imgenex, San Diego, CA).
Transfections and luciferase assays. Transfections in 293T cells were per-
formed with FuGENE 6 (Roche) according to the manufacturer’s instructions.
For siRNA transfections, cells were transfected with 60 pmol of control or Ubc13
siRNA using Lipofectamine 2000 (Invitrogen). Plasmid DNAs were transfected
using FuGENE 6 the next day after siRNA transfections. Cells were harvested 4
days after the siRNA transfection. Luciferase assays were performed by using a
dual luciferase assay kit (Promega, Madison, WI). All luciferase transfections
included the Renilla luciferase reporter pRL-tk to normalize for transfection
efficiency. Transfections for luciferase assays were performed in triplicate, and
error bars represent the standard errors of the means. Transient transfections in
MEFs were performed using FuGENE HD (Roche) according to the manufac-
EMSA. The NF-?B electrophoretic mobility shift assay (EMSA) was done as
described previously (22, 48). The Oct-1 EMSA probe was generated by anneal-
ing the following oligonucleotides: forward 5?-TGTCGAATGCAAATCACT
AGAA and reverse 5?-TTCTAGTGAT. The annealed oligonucleotides were
labeled with [32P]dTTP in a fill-in reaction with Klenow fragment (Promega).
Nuclear extract (4 ?g) was incubated with buffer containing 1 mM dithiothreitol,
1 ?g poly(dI-dC), dialysis buffer (25 mM HEPES, pH 7.9, 10% glycerol, 100 mM
KCl, and 0.1 mM EDTA), and32P-labeled probe for 15 min. The reaction was
terminated by the addition of 5? loading dye, and the reaction mixture was run
on 5% polyacrylamide gels in 0.25? Tris-borate-EDTA buffer, dried under
vacuum, and subjected to autoradiography.
Generation of Ubc13?/?MEFs. Primary MEFs from control and Ubc13fl/fl
embryos were immortalized with simian virus 40 large T antigen. T-antigen
expression was confirmed by reverse transcription (RT)-PCR. Control and
Ubc13fl/flimmortalized MEFs were infected with Cre-expressing recombinant
retroviruses as described below.
Retroviral infections. Retrovirus-mediated transfer of Cre into MEFs was
performed by transfecting pMRXP-Cre into the Plat-E packaging cell line (38).
For all other retroviral infections, retroviral vectors (pCLXSN, pCLXSN-Tax,
pSuppressor Retro-GFP siRNA, and pSuppressor Retro-Ubc13 siRNA) were
transfected into 293T cells together with pCL-Ampho and vesicular stomatitis
virus glycoprotein. The supernatants were filtered and used to infect MEFs or
Jurkat or C8166 cells in the presence of Polybrene (8 ?g/ml). Jurkat and C8166
cells were centrifuged at 1,800 rpm for 45 min after resuspension in viral super-
natant, to increase the infection efficiency. After 72 h, cells were selected in
medium containing G418 (Invitrogen) to obtain stable, bulk cell lines. Stable
Jurkat and C8166 cell lines were selected with 1 ?g/ml G418. Stable MEFs were
selected with 0.4 ?g/ml G418.
RT-PCR. RT-PCR was done as described previously (24). Total RNA was
obtained from cells by using an RNeasy kit (QIAGEN, Valencia, CA) and
converted to cDNA by using a first-strand cDNA synthesis kit (Roche). The
following sets of primers were used to amplify gene products for PCRs: glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH) (263 bp) forward-5?-CCACA
GTCCATGCCATCAC and reverse-5?-GCTTCACCACCTTCTTGATG; Tax
(429 bp) forward-5?-CGGATACCCAGTCTACGTC and reverse-5?-GAGGTA
CATGCAGACAACGG; IL-2 (470 bp) forward-5?-GTACAGGATGCAACTC
CTGTC and reverse-5?-CAAGTTAGTGTTGAGATGATGC; IL-2R? (818 bp)
forward-5?-GATGGATTCATACCTGCTGATG and reverse-5?-CTAGATTGT
TCTTCTACTCTTCC; IL-6 (460 bp) forward-5?-GACTTCACAGAGGATACC
ACTC and reverse-5?-GTCCTTAGCCACTCCTTCTG; and A20 (560 bp)
forward-5?-GACAGAAGTGTCCAGGCTTC and reverse-5?-GTGCTGGCTG
Coimmunoprecipitation and ubiquitination assays. Ubiquitination assays
were performed essentially as described previously (55). In 293T cells, ubiquiti-
nation assays were performed by transfecting pCMV4-Tax and Flag-TRAF6
together with HA-Ub, HA-Ub K63-only, or HA-Ub K48-only plasmids. Approx-
imately 40 h after transfection, cells were lysed in radioimmunoprecipitation
assay (RIPA) buffer and subjected to immunoprecipitation with anti-Tax or
anti-FLAG monoclonal antibodies. The agarose beads were washed three times
with RIPA buffer, followed by an additional wash with RIPA buffer containing 1
M urea. Proteins were eluted from the beads, subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and detected by immunoblotting with
anti-HA monoclonal antibody. For Tax ubiquitination assays in MEFs, Tax was
immunoprecipitated with anti-Tax and immunoblotted with an antiubiquitin
Western blotting. Western blotting was done as described previously (22).
Cells were lysed in RIPA buffer, and the lysates were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose
membranes, blocked in 5% milk, incubated with appropriate primary and sec-
ondary antibodies, and detected by Western Lightning enhanced chemilumines-
cence reagent (Perkin Elmer, Boston, MA).
Tax polyubiquitination is predominantly K63-linked. Al-
though several reports have documented Tax ubiquitination,
the exact biochemical nature of the types of ubiquitin linkages
has remained elusive. Ubiquitin molecules harbor 7 lysine res-
idues, each of which may mediate polyubiquitin linkages; how-
ever, the best characterized are K48- and K63-linked poly-
ubiquitin chains. Polyubiquitin chains linked to one another via
13736 SHEMBADE ET AL.J. VIROL.
K48 target proteins for degradation by the proteasome (4). In
contrast, the K63-linked polyubiquitin chains do not target
proteins for degradation but rather modulate the subcellular
locations or signaling functions of the ubiquitinated proteins
(5, 19). Thus, we performed Tax ubiquitination assays with
either wild-type ubiquitin, a ubiquitin variant with all of the
lysines except for lysine 63 mutated to arginine (K63-only), or
a ubiquitin variant with all of the lysines except for lysine 48
mutated to arginine (K48-only). We transfected 293T cells with
a Tax expression plasmid together with each of the HA-tagged
ubiquitin constructs and performed ubiquitination assays. As a
control for K63 ubiquitination, we also transfected Flag-
TRAF6 with each of the ubiquitin constructs. In agreement
with the results of published studies (6, 34, 39, 41), polyubiq-
uitination of Tax was readily detected in cells expressing the
wild-type ubiquitin (Fig. 1A). More importantly, Tax ubiqui-
tination also occurred efficiently in cells expressing K63-only
ubiquitin, but poorly in cells expressing the K48-only ubiquitin
(Fig. 1A). As expected, TRAF6 was also ubiquitinated, and the
polyubiquitin chains were mainly K63-linked (Fig. 1A). These
results suggest that Tax ubiquitination is predominantly com-
posed of K63-linked polyubiquitin chains, although we cannot
rule out polyubiquitin chains linked via lysines independent of
K48 and K63.
FIG. 1. Tax polyubiquitination is K63-linked. (A) 293T cells were transfected with 2 ?g pCMV4-Tax or 1 ?g Flag-TRAF6 together with 0.5 ?g
of HA-Ub, HA-Ub K48-only, or HA-Ub K63-only. After 36 h, cells were lysed and immunoprecipitated (IP) with either anti-Tax or anti-Flag
antibodies, followed by immunoblotting (IB) with anti-HA. Lysates were examined for Tax and TRAF6 expression by immunoblotting with
anti-Tax and anti-Flag, respectively. ?, pCMV4 only. (B) 293T cells were transfected with pCMV4, 0.5 ?g of HA-Ub together with 2 ?g of
pCMV4-Tax, pCMV4-Tax M22, or pCMV4-Tax M47. After 36 h, cells were lysed and immunoprecipitated with anti-HA, followed by immuno-
blotting with anti-Tax. Ig H.C., immunoglobulin heavy chain; Ig L.C., immunoglobulin light chain; ?, pCMV4 only. (C) 293T cells were transfected
with ?B-TATA luciferase (0.1 ?g), pRL-tk (0.01 ?g), and pCMV4-Tax (1 ?g), HA-Ub (0.25 ?g), or HA-Ub K63-only (0.25 ?g) as indicated. Cells
were harvested after 36 h and dual luciferase assays were performed. NF-?B induction (n-fold) for each sample compared to vector alone was
calculated. Statistical analysis was performed by one-way analysis of variance, followed by the Tukey-Kramer test for multiple comparisons (?, P
? 0.01; ??, P ? 0.001). Tax expression in lysates was determined by immunoblotting with anti-Tax. Molecular sizes are shown to the left of the
panels. Luc., luciferase; ?, anti.
VOL. 81, 2007Tax REQUIRES Ubc13 FOR NF-?B ACTIVATION 13737
To confirm that the polyubiquitinated Tax was indeed Tax
and not an associated protein, we performed a reciprocal im-
munoprecipitation in which HA-Ub was immunoprecipitated,
followed by immunoblotting with Tax antibody. 293T cells
were transfected with HA-Ub together with wild-type Tax, Tax
M22 (a point mutant deficient in NF-?B activation), and Tax
M47 (a point mutant deficient in CREB activation) (49).
Monoubiquitinated forms of Tax, Tax M22, and Tax M47 were
detected, although lower levels of monoubiquitinated Tax M22
were observed (Fig. 1B). A ladder of bands representing Tax
and Tax M47 ubiquitin adducts were also detected; however,
polyubiquitinated M22 was not observed, in agreement with
the results of another study (Fig. 1B) (6). Therefore, these
results indicate that Tax is polyubiquitinated and that Tax M22
displays a defect in polyubiquitination.
Next, we examined a potential functional role for K63-linked
ubiquitin chains in Tax-mediated NF-?B activation. 293T cells
were transfected with Tax in combination with either the
HA-Ub or HA-Ub K63-only plasmids, and NF-?B luciferase
assays were performed. As expected, Tax expression resulted
in potent activation of the NF-?B luciferase reporter (Fig. 1C).
Transfection of HA-Ub and HA-Ub K63-only plasmids led to
significant increases in Tax-mediated NF-?B activation, al-
though they had little effect on NF-?B activation in the absence
of Tax (Fig. 1C). Immunoblotting confirmed that Tax was
expressed at similar levels in transfected cells (Fig. 1C). Thus,
ubiquitination, and specifically K63-linked polyubiquitination,
strongly promotes the Tax activation of NF-?B.
The E2 ubiquitin-conjugating enzyme Ubc13 is required for
Tax ubiquitination. Ubiquitination is a reversible covalent
modification that occurs on lysine residues in target proteins.
Ubiquitination occurs in an ordered three-step process that is
dependent on a ubiquitin-activating enzyme (E1), ubiquitin-
conjugating enzymes (E2), and ubiquitin ligases (E3) that pro-
vide specificity for ubiquitination (27). A number of E3 ligases,
including TRAF6, RNF5, RNF8, and CHFR (3, 9, 33, 43),
have been identified that require Ubc13 to catalyze the forma-
tion of K63-linked polyubiquitin chains. Because Tax ubiqui-
tination was predominantly K63-linked, we examined the role
of Ubc13 in Tax ubiquitination. We performed Tax ubiqui-
tination assays in the presence of control or Ubc13-specific
siRNA. As expected, Tax was polyubiquitinated in the pres-
ence of HA-Ub or HA-Ub K63-only and control siRNA (Fig.
2). However, knockdown of Ubc13 with siRNA led to a signif-
icant reduction in Tax ubiquitination (Fig. 2) suggesting that
Ubc13 plays an important role in Tax ubiquitination.
Tax interacts with Ubc13. Since Ubc13 was required for Tax
ubiquitination, we next examined if Tax interacted with Ubc13.
293T cells were transfected with wild-type Tax, Tax M22, and
Tax M47. Cells expressing Tax and Tax mutants were then
subjected to a coimmunoprecipitation assay for interaction
with endogenous Ubc13. Surprisingly, wild-type Tax and Tax
M47, but not Tax M22, interacted with Ubc13 (Fig. 3A). We
also performed the reciprocal immunoprecipitation where Tax
antibody was used for the immunoprecipitation, followed by
immunoblotting with anti-Ubc13. Again, Tax and Tax M47, but
not Tax M22, were found to interact with endogenous Ubc13
(Fig. 3B). The lack of binding of Tax M22 to Ubc13 may
explain why M22 exhibits a defect in polyubiquitination (Fig.
1). Tax M22 was also previously shown to be defective for
binding to NEMO (23). The interaction of Tax and Ubc13 was
also confirmed in the HTLV-1-transformed cell lines C8166
(Fig. 3C) and MT-2 (data not shown). Given that E2 enzymes
mainly interact with their cognate E3 ligases, these results
indicate that Tax is likely tightly bound to an E3 ligase complex
of which Ubc13 is a component.
Ubc13 is required for Tax activation of NF-?B. Prior studies
have relied on Tax point mutants to establish a link between
Tax ubiquitination and NF-?B activation. Since modification
of Tax by mutation or deletion may potentially impair Tax
function nonspecifically, it was important to confirm the role of
Tax ubiquitination in NF-?B activation without manipulating
Tax sequences. Therefore, we used siRNA to knock down
Ubc13 expression and examined Tax activation of both the
canonical and noncanonical NF-?B pathways. 293T cells were
transfected with Tax together with control scrambled siRNA or
Ubc13 siRNA. Knockdown of Ubc13 expression by siRNA
FIG. 2. Tax ubiquitination is dependent on Ubc13. 293T cells were
transfected with control siRNA or Ubc13 siRNA. The next day, the
same cells were transfected with vector alone (first and fourth lanes) or
pCMV4-Tax (2 ?g) together with either 0.5 ?g of HA-Ub or HA-Ub
K63-only. After 36 h, cells were lysed and immunoprecipitated (IP)
with anti-Tax, followed by immunoblotting (IB) with anti-HA. Lysates
were examined for Tax and Ubc13 expression. Molecular sizes are
shown to the left of the panel. ?, anti; cont., control; ?, pCMV4 only.
FIG. 3. Tax interacts with Ubc13. (A) 293T cells were transfected
with 2 ?g vector (first lane), pCMV4-Tax, pCMV4-Tax M22, or
pCMV4-Tax M47. After 36 h, cells were lysed and immunoprecipitated
(IP) with anti-Ubc13, followed by immunoblotting (IB) with anti-Tax.
Lysates were examined for Tax expression. (B) 293T cells were trans-
fected as described for panel A. Lysates were immunoprecipitated with
anti-Tax, followed by immunoblotting with anti-Ubc13. (C) C8166 cells
(1 ? 107) were lysed and immunoprecipitated with control immuno-
globulin (Ig), anti-Ubc13, or anti-NEMO, followed by immunoblotting
with anti-Tax. ?, anti; ?, pCMV4 only.
13738 SHEMBADE ET AL.J. VIROL.
resulted in defective NF-?B activation by Tax as assessed by
phosphorylation of I?B? (Fig. 4A) and NF-?B DNA binding
(data not shown). It has been previously shown that Tax is a
potent activator of p100 processing to p52 in the noncanonical
NF-?B pathway (53). Therefore, we examined if Ubc13 was
required for Tax-mediated p100 processing. Indeed, knock-
down of Ubc13 completely impaired the ability of Tax to in-
duce p100 processing (Fig. 4B). Thus, Ubc13 is essential for
Tax activation of both the canonical and noncanonical NF-?B
Since T lymphocytes are the natural target of HTLV-1 in-
fection in vivo, we extended our studies to include T cells. To
knock down Ubc13 expression in T cells, we infected Jurkat T
cells with retroviral vectors expressing a control GFP siRNA or
Ubc13 siRNA. Stable, bulk cell lines were derived by selection
in medium containing G418. The absence of Ubc13 in the
Ubc13 siRNA-expressing cell line was confirmed by immuno-
blotting (Fig. 4C). The tax gene was introduced into these cells
by retroviral gene transfer to determine the effect of Ubc13
knockdown on Tax activation of NF-?B in T cells. Importantly,
Tax was expressed in both control and Ubc13 knockdown
Jurkat cells as determined by RT-PCR (Fig. 4E). Jurkat cells
lacking Ubc13 expression were impaired in Tax-mediated
NF-?B DNA binding (Fig. 4D). A control Oct-1 EMSA dem-
onstrated similar DNA binding in all of the nuclear extracts
(Fig. 4D). Thus, Ubc13 is required for Tax-mediated NF-?B
activation in T cells. Moreover, the induction by Tax of T-cell-
specific NF-?B-dependent target genes, including those for
IL-2 and IL-2R?, was abolished in the absence of Ubc13 ex-
pression (Fig. 4E). To determine the role of Ubc13 in NF-?B
activation in a Tax-expressing HTLV-1-transformed cell line,
we infected C8166 cells with retroviral vectors expressing con-
trol GFP or Ubc13 siRNA. Ubc13 knockdown in C8166 cells
was confirmed by immunoblotting (Fig. 4F). As expected, con-
stitutive NF-?B DNA binding was observed in C8166 GFP
siRNA control cells; however, siRNA-mediated knockdown of
Ubc13 in C8166 cells abolished NF-?B DNA binding (Fig. 4F).
Oct-1 DNA binding was similar in both nuclear extracts (Fig.
4F). Ubc13 is therefore required for NF-?B activation in the
context of a Tax-expressing HTLV-1-transformed cell line.
We next examined Tax activation of NF-?B in Ubc13-defi-
cient MEFs. Primary MEFs were obtained from Ubc13?/?and
Ubc13fl/flembryos engineered with loxP sites flanking exons 2
and 4 (56). Retrovirus-mediated delivery of Cre recombinase
in Ubc13?/?and Ubc13fl/fleffectively eliminated Ubc13 ex-
pression only in Ubc13fl/flMEFs (Fig. 5A). Consistent with the
results of previous studies, IL-1 and TNF-? treatment led to
I?B? phosphorylation and degradation (Fig. 5B) and concom-
itant NF-?B DNA binding in Ubc13fl/flMEFs (Fig. 5C) (12,
56). However, Tax was completely impaired in I?B? phosphor-
ylation and degradation (Fig. 5B) and NF-?B DNA binding in
Ubc13fl/flMEFs (Fig. 5C). Reconstitution of Ubc13-deficient
MEFs with wild-type Ubc13, but not a Ubc13 mutant (Ubc13
C87A) harboring a Cys3Ala mutation in its active site, res-
cued Tax-mediated NF-?B activation, as shown by an NF-?B
EMSA (Fig. 5D). The ubiquitination of Tax was also impaired
FIG. 4. Tax requires Ubc13 for NF-?B activation. (A) 293T cells were transfected with control siRNA or Ubc13 siRNA. The next day, the same
cells were transfected with 2 ?g of vector (first lane) or pCMV4-Tax as indicated above the panel. After 36 h, cells were lysed and lysates were
subjected to immunoblotting (IB) with anti-phospho-I?B?, anti-Tax, and anti-Ubc13. (B) 293T cells were transfected with control or Ubc13
siRNA. The next day, the same cells were transfected with pCMV4-p100 (0.25 ?g) and pCMV4-Tax (2 ?g) as indicated above the panel. After
36 h, lysates were immunoblotted with anti-p100, anti-Tax, and anti-Ubc13. (C) Jurkat GFP siRNA and Jurkat Ubc13 siRNA stable cell lines were
lysed and subjected to immunoblotting with anti-Ubc13 and anti-?-actin. (D) Nuclear extracts from Jurkat GFP siRNA and Jurkat Ubc13 siRNA
stable cell lines infected with pCLXSN (first and third lanes) or pCLXSN-Tax were subjected to NF-?B and Oct-1 EMSA. (E) RNA from Jurkat
GFP siRNA and Jurkat Ubc13 siRNA stable cell lines infected with pCLXSN (first and third lanes) or pCLXSN-Tax (second and fourth lanes)
was used for RT-PCR to examine mRNA levels of Tax, IL-2, IL-2R?, and GAPDH. ?, present; ?, absent. (F) C8166 GFP siRNA and C8166
Ubc13 siRNA stable cell lines were lysed and subjected to immunoblotting with anti-Ubc13 and anti-?-actin (top panels). Nuclear extracts were
used for NF-?B and Oct-1 EMSA (lower panels). ?, anti; cont., control; ?, pCMV4 only.
VOL. 81, 2007 Tax REQUIRES Ubc13 FOR NF-?B ACTIVATION13739
in Ubc13 knockout MEFs (Fig. 5E), consistent with a role for
Tax ubiquitination regulating NF-?B activation. Finally, Tax
induction of NF-?B-dependent target genes was examined by
RT-PCR. Tax-mediated induction of A20 and IL-6 was defec-
tive in Ubc13fl/flMEFs (Fig. 5F). Therefore, in MEFs lacking
Ubc13, Tax is completely impaired in the ubiquitination and
activation of NF-?B.
Ubc13 is required for Tax interaction with NEMO. To fur-
ther explore the mechanistic link between Tax ubiquitination
and NF-?B activation, we examined the binding of Tax and
NEMO in the absence of Ubc13 expression. First, we exam-
ined the binding of NEMO with a Tax ubiquitination-defective
mutant that has all 10 lysine residues mutated to arginines (Tax
K10R) (6). Tax interaction with NEMO was observed with
wild-type Tax, but not the Tax K10R mutant (Fig. 6A). Tax
binding to NEMO was also examined in the absence of Ubc13
expression. Tax interaction with endogenous NEMO occurred
in the presence of control siRNA, but not Ubc13 siRNA (Fig.
6B). Therefore, Tax requires Ubc13 for interaction with
The HTLV-1 Tax oncoprotein is a potent intracellular acti-
vator of NF-?B by promotion of the constitutive activation of
IKK. The stable ubiquitination of Tax has emerged as an
important regulator of Tax-mediated NF-?B activation (39).
However, previous studies have relied on Tax point mutants to
demonstrate the importance of ubiquitination in NF-?B acti-
vation. In this study, we have determined that Tax polyubiq-
uitin chains are primarily K63 linked and that the E2 ubiquitin-
conjugating enzyme Ubc13 is essential for Tax ubiquitination.
Tax interacts with Ubc13 both in transfected cells and in
HTLV-1-transformed cell lines. Moreover, Tax requires
FIG. 5. Tax-mediated NF-?B activation is impaired in Ubc13?/?MEFs. (A) Ubc13?/?and Ubc13fl/flMEFs infected with Cre-expressing
retroviruses were lysed and subjected to immunoblotting (IB) with anti-Ubc13 and anti-?-actin. (B and C) Ubc13?/?and Ubc13fl/flMEFs
expressing Cre were either stimulated with IL-1 (10 ng/ml) or TNF-? (20 ng/ml) for 30 min or infected with Tax-expressing retroviruses. ?, no
stimulation. (B) Lysates were subjected to immunoblotting with anti-I?B?, anti-phospho-I?B? and anti-Tax. (C) Nuclear extracts were used for
NF-?B and Oct-1 EMSA. (D) Ubc13fl/flMEFs expressing Tax were transiently transfected with pCMV4, HA-Ubc13, or HA-Ubc13 C87A. After
36 h, nuclear extracts were subjected to an NF-?B EMSA. The expression of ectopic Ubc13 was determined in cytoplasmic (cyt.) and nuclear (nuc.)
extracts by immunoblotting with anti-HA. (E) Ubc13?/?and Ubc13fl/flMEFs expressing Cre were infected with retroviruses expressing Tax (?)
or empty vector (?). After 72 h, cells were lysed and immunoprecipitated with anti-Tax, followed by immunoblotting with anti-ubiquitin. Lysates
were examined for Tax expression. (F) Ubc13?/?and Ubc13fl/flMEFs expressing Cre were infected with Tax as described for panel D. After 72 h,
RNA was harvested and subjected to RT-PCR analysis to examine the expression of A20, IL-6, Tax, and GAPDH. ?, anti.
FIG. 6. Tax requires Ubc13 to interact with NEMO. (A) 293T cells
were transfected with 2 ?g of pSG-5-Tax or pSG-5-Tax K10R and
HA-NEMO (50 ng). After 36 h, cells were lysed and subjected to
immunoprecipitations with anti-HA followed by immunoblotting with
anti-Tax. Lysates were examined for Tax and NEMO expression with
anti-Tax and anti-HA, respectively. (B) 293T cells were transfected
with control (cont.) or Ubc13 siRNA. The next day, the same cells
were transfected with pCMV4-Tax (2 ?g) or empty vector (first lane).
After 36 h, cells were lysed and immunoprecipitated with anti-NEMO,
followed by immunoblotting with anti-Tax. Lysates were examined for
Ubc13 expression. ?, anti; ?, absent.
13740SHEMBADE ET AL.J. VIROL.
Ubc13 for the activation of NF-?B and the induction of NF-
?B-dependent target genes, including those for IL-2 and its
high-affinity receptor IL-2R? in T cells. Finally, in the absence
of Ubc13, Tax is impaired in NEMO binding, suggesting that
the role of Tax ubiquitination is to mediate protein-protein
The findings from this study suggest that Ubc13 is essential
for the activation of NF-?B by Tax, but not by IL-1 or TNF-?
stimulation. Ubc13 has previously been shown to be dispens-
able for IL-1- and TNF-?-mediated NF-?B activation in MEFs
(56). Furthermore, the processing of p100 to p52 was normal in
Ubc13-deficient B cells stimulated with ?-CD40 or B-cell-ac-
tivating factor of the TNF family (56). Thus, the mechanisms
used by Tax to activate NF-?B are clearly different from the
IL-1R and TNF receptor signaling pathways, although NEMO
is required for NF-?B activation by Tax and cytokine stimula-
tion (20, 58). In Tax-mediated NF-?B activation, the role of
Ubc13 is to facilitate Tax K63-linked polyubiquitination (Fig. 2
and 5), which in turn mediates the interaction with NEMO
(Fig. 6). The exact mechanism by which Tax stimulates IKK
activity is poorly understood but may involve upstream kinases
such as transforming growth factor ?-activated kinase 1 (52).
Interestingly, the Tax M22 mutant defective for NF-?B activa-
tion is unable to interact with either NEMO (23) or Ubc13
(Fig. 3), suggesting that an association of Tax with NEMO
and/or Ubc13 is critical for NF-?B activation. Since the fusion
of Tax M22 with NEMO restores NF-?B activation (54), it will
be interesting to determine if the fusion of Ubc13 to Tax M22
will similarly activate NF-?B.
Our results are not in agreement with those of a recent study
demonstrating that Tax activation of NF-?B is independent of
Ubc13 (15). However, the conclusions by Gohda et al. were
derived from a single experiment, an NF-?B luciferase assay
with siRNA-mediated knockdown of Ubc13 in 293T cells (15).
It is unclear to us why our results are inconsistent with the
findings by Gohda et al. Our findings in four different cell
types, 293T, Ubc13?/?MEFs, Jurkat, and C8166 cells, using a
variety of different assays, consistently support a role for Ubc13
in Tax-mediated NF-?B activation. Furthermore, we have pro-
vided genetic evidence that Tax requires Ubc13 for the induc-
tion of NF-?B-dependent genes, such as those for IL-6 and
IL-2. Consistent with the results of our studies, Kfoury et al.
recently demonstrated that Tax undergoes K63-linked
Although previous studies have established Tax ubiquitina-
tion, the biochemical nature of the polyubiquitin chain link-
ages has remained elusive. Here, we describe Tax polyubiqui-
tination as predominantly K63 linked. However, our findings
do not rule out the ubiquitination of Tax through other types
of ubiquitin linkages. K63-linked ubiquitination has emerged
as an important regulator of a variety of different biological
processes, including receptor trafficking and protein-protein
interactions. The Kaposi’s sarcoma-associated herpesvirus K3
gene product promotes the internalization of cell surface ma-
jor histocompatibility complex class I molecules by K63-linked
ubiquitination that is dependent on Ubc13 (10). Similarly,
TRAF6-dependent K63-linked ubiquitination of the nerve
growth factor receptor TrkA and neurotrophin receptor inter-
acting factor regulates the trafficking and localization of these
proteins (13, 14). K63-linked ubiquitination may also regulate
protein function, as recently demonstrated for interferon reg-
ulatory factor 7, which exhibits enhanced transcriptional activ-
ity when ubiquitinated (28). In addition, p53 localization and
activity are regulated by Ubc13- and K63-linked ubiquitination
(32). To our knowledge, Tax is the first viral oncoprotein iden-
tified that is regulated by K63-linked ubiquitination.
K63-linked polyubiquitin chains may function as scaffolds
that promote the assembly of complexes containing kinases
and other signaling proteins (1). Thus, Tax polyubiquitination
may nucleate a signaling complex that promotes the activation
of IKK. Indeed, our results indicate that NEMO is likely an
essential component of this complex. There are likely addi-
tional, yet-to-be-identified proteins within this complex, possi-
bly including an E3 ligase specific for Tax. TRAF2, TRAF6,
RNF5, RNF8, and CHFR are all E3 ligases capable of cata-
lyzing K63-linked ubiquitination that is dependent on Ubc13
(3, 9, 17, 33, 43). Our preliminary data indicate that TRAF6 is
not required for Tax-mediated NF-?B activation; therefore,
TRAF6 may not serve as the Tax E3 ligase (data not shown).
A more remote possibility is that Tax itself functions as an E3
ligase since Tax interacts with Ubc13. However, it is not clear
if Tax interaction with Ubc13 is direct, and Tax lacks a con-
served domain typical of E3 ligases, such as HECT (46) or
RING (42). Future studies will more precisely determine the
requirements for Tax ubiquitination and NF-?B activation.
We thank Shao-Cong Sun (University of Texas M. D. Anderson
Cancer Center), Warner Greene (UCSF Gladstone), James Chen
(University of Texas Southwestern Medical Center), Claudine Pique
(Institut Cochin), Khaled Tolba (University of Miami), Matt Morrison
(Cell Signaling Technologies), and Toshio Kitamura (University of
Tokyo) for reagents.
These studies were supported in part by Public Health Service grant
RO1 CA99926 to E.W.H. from the National Cancer Institute.
1. Adhikari, A., M. Xu, and Z. J. Chen. 2007. Ubiquitin-mediated activation of
TAK1 and IKK. Oncogene 26:3214–3226.
2. Beinke, S., and S. C. Ley. 2004. Functions of NF-?B1 and NF-?B2 in immune
cell biology. Biochem. J. 382:393–409.
3. Bothos, J., M. K. Summers, M. Venere, D. M. Scolnick, and T. D. Halazonetis.
2003. The Chfr mitotic checkpoint protein functions with Ubc13-Mms2 to form
Lys63-linked polyubiquitin chains. Oncogene 22:7101–7107.
4. Chau, V., J. W. Tobias, A. Bachmair, D. Marriott, D. J. Ecker, D. K. Gonda,
and A. Varshavsky. 1989. A multiubiquitin chain is confined to specific lysine
in a targeted short-lived protein. Science 243:1576–1583.
5. Chen, Z. J. 2005. Ubiquitin signalling in the NF-?B pathway. Nat. Cell Biol.
6. Chiari, E., I. Lamsoul, J. Lodewick, C. Chopin, F. Bex, and C. Pique. 2004.
Stable ubiquitination of human T-cell leukemia virus type 1 Tax is required
for proteasome binding. J. Virol. 78:11823–11832.
7. Claudio, E., K. Brown, S. Park, H. Wang, and U. Siebenlist. 2002. BAFF-
induced NEMO-independent processing of NF-?B2 in maturing B cells. Nat.
8. Deng, L., C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter, C.
Pickart, and Z. J. Chen. 2000. Activation of the I?B kinase complex by
TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a
unique polyubiquitin chain. Cell 103:351–361.
9. Didier, C., L. Broday, A. Bhoumik, S. Israeli, S. Takahashi, K. Nakayama,
S. M. Thomas, C. E. Turner, S. Henderson, H. Sabe, and Z. Ronai. 2003.
RNF5, a RING finger protein that regulates cell motility by targeting paxillin
ubiquitination and altered localization. Mol. Cell. Biol. 23:5331–5345.
10. Duncan, L. M., S. Piper, R. B. Dodd, M. K. Saville, C. M. Sanderson, J. P.
Luzio, and P. J. Lehner. 2006. Lysine-63-linked ubiquitination is required for
endolysosomal degradation of class I molecules. EMBO J. 25:1635–1645.
11. Fu, D. X., Y. L. Kuo, B. Y. Liu, K. T. Jeang, and C. Z. Giam. 2003. Human
T-lymphotropic virus type I Tax activates I?B kinase by inhibiting I?B ki-
nase-associated serine/threonine protein phosphatase 2A. J. Biol. Chem.
VOL. 81, 2007 Tax REQUIRES Ubc13 FOR NF-?B ACTIVATION 13741
12. Fukushima, T., S. Matsuzawa, C. L. Kress, J. M. Bruey, M. Krajewska, S.
Lefebvre, J. M. Zapata, Z. Ronai, and J. C. Reed. 2007. Ubiquitin-conjugat-
ing enzyme Ubc13 is a critical component of TNF receptor-associated factor
(TRAF)-mediated inflammatory responses. Proc. Natl. Acad. Sci. USA 104:
13. Geetha, T., J. Jiang, and M. W. Wooten. 2005. Lysine 63 polyubiquitination
of the nerve growth factor receptor TrkA directs internalization and signal-
ing. Mol. Cell 20:301–312.
14. Geetha, T., R. S. Kenchappa, M. W. Wooten, and B. D. Carter. 2005.
TRAF6-mediated ubiquitination regulates nuclear translocation of NRIF,
the p75 receptor interactor. EMBO J. 24:3859–3868.
15. Gohda, J., M. Irisawa, Y. Tanaka, S. Sato, K. Ohtani, J. Fujisawa, and
J. Inoue. 2007. HTLV-1 Tax-induced NF-?B activation is independent of
Lys-63-linked-type polyubiquitination. Biochem. Biophys. Res. Commun.
16. Grant, C., K. Barmak, T. Alefantis, J. Yao, S. Jacobson, and B. Wigdahl.
2002. Human T cell leukemia virus type I and neurologic disease: events in
bone marrow, peripheral blood, and central nervous system during normal
immune surveillance and neuroinflammation. J. Cell. Physiol. 190:133–159.
17. Habelhah, H., S. Takahashi, S. G. Cho, T. Kadoya, T. Watanabe, and Z.
Ronai. 2004. Ubiquitination and translocation of TRAF2 is required for
activation of JNK but not of p38 or NF-?B. EMBO J. 23:322–332.
18. Hacker, H., and M. Karin. 2006. Regulation and function of IKK and
IKK-related kinases. Sci. STKE 2006:re13.
19. Haglund, K., and I. Dikic. 2005. Ubiquitylation and cell signaling. EMBO J.
20. Harhaj, E. W., L. Good, G. Xiao, M. Uhlik, M. E. Cvijic, I. Rivera-Walsh,
and S. C. Sun. 2000. Somatic mutagenesis studies of NF-?B signaling in
human T cells: evidence for an essential role of IKK? in NF-?B activation by
T-cell costimulatory signals and HTLV-I Tax protein. Oncogene 19:1448–
21. Harhaj, E. W., and N. S. Harhaj. 2005. Mechanisms of persistent NF-?B
activation by HTLV-I Tax. IUBMB Life 57:83–91.
22. Harhaj, E. W., N. S. Harhaj, C. Grant, K. Mostoller, T. Alefantis, S. C. Sun,
and B. Wigdahl. 2005. Human T cell leukemia virus type I Tax activates
CD40 gene expression via the NF-?B pathway. Virology 333:145–158.
23. Harhaj, E. W., and S. C. Sun. 1999. IKK? serves as a docking subunit of the
I?B kinase (IKK) and mediates interaction of IKK with the human T-cell
leukemia virus Tax protein. J. Biol. Chem. 274:22911–22914.
24. Harhaj, N. S., B. Janic, J. C. Ramos, W. J. Harrington, Jr., and E. W.
Harhaj. 2007. Deregulated expression of CD40 ligand in HTLV-I infection:
distinct mechanisms of downregulation in HTLV-I-transformed cell lines
and ATL patients. Virology 362:99–108.
25. Harhaj, N. S., S. C. Sun, and E. W. Harhaj. 2007. Activation of NF-?B by the
human T cell leukemia virus type I Tax oncoprotein is associated with
ubiquitin-dependent relocalization of I?B kinase. J. Biol. Chem. 282:4185–
26. Hayden, M. S., and S. Ghosh. 2004. Signaling to NF-?B. Genes Dev. 18:
27. Hershko, A., and A. Ciechanover. 1998. The ubiquitin system. Annu. Rev.
28. Huye, L. E., S. Ning, M. Kelliher, and J. S. Pagano. 2007. Interferon regu-
latory factor 7 is activated by a viral oncoprotein through RIP-dependent
ubiquitination. Mol. Cell. Biol. 27:2910–2918.
29. Jeang, K. T., C. Z. Giam, F. Majone, and M. Aboud. 2004. Life, death, and
Tax: role of HTLV-I oncoprotein in genetic instability and cellular transfor-
mation. J. Biol. Chem. 279:31991–31994.
30. Jin, D. Y., V. Giordano, K. V. Kibler, H. Nakano, and K. T. Jeang. 1999. Role
of adapter function in oncoprotein-mediated activation of NF-?B. Human
T-cell leukemia virus type I Tax interacts directly with IKK?. J. Biol. Chem.
31. Kfoury, Y., R. Nasr, A. Favre-Bonvin, M. El-Sabban, N. Renault, M. L.
Giron, N. Setterblad, H. E. Hajj, E. Chiari, A. G. Mikati, O. Hermine, A.
Saib, H. de The, C. Pique, and A. Bazarbachi. 24 September 2007. Ubiqui-
tylated Tax targets and binds the IKK signalosome at the centrosome. On-
32. Laine, A., I. Topisirovic, D. Zhai, J. C. Reed, K. L. Borden, and Z. Ronai.
2006. Regulation of p53 localization and activity by Ubc13. Mol. Cell. Biol.
33. Lamothe, B., A. Besse, A. D. Campos, W. K. Webster, H. Wu, and B. G.
Darnay. 2007. Site-specific Lys-63-linked tumor necrosis factor receptor-
associated factor 6 auto-ubiquitination is a critical determinant of I?B kinase
activation. J. Biol. Chem. 282:4102–4112.
34. Lamsoul, I., J. Lodewick, S. Lebrun, R. Brasseur, A. Burny, R. B. Gaynor,
and F. Bex. 2005. Exclusive ubiquitination and sumoylation on overlapping
lysine residues mediate NF-?B activation by the human T-cell leukemia virus
Tax oncoprotein. Mol. Cell. Biol. 25:10391–10406.
35. Lomaga, M. A., W. C. Yeh, I. Sarosi, G. S. Duncan, C. Furlonger, A. Ho, S.
Morony, C. Capparelli, G. Van, S. Kaufman, A. van der Heiden, A. Itie, A.
Wakeham, W. Khoo, T. Sasaki, Z. Cao, J. M. Penninger, C. J. Paige, D. L.
Lacey, C. R. Dunstan, W. J. Boyle, D. V. Goeddel, and T. W. Mak. 1999.
TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40,
and LPS signaling. Genes Dev. 13:1015–1024.
36. Marriott, S. J., and O. J. Semmes. 2005. Impact of HTLV-I Tax on cell cycle
progression and the cellular DNA damage repair response. Oncogene 24:
37. Mori, N., Y. Yamada, S. Ikeda, Y. Yamasaki, K. Tsukasaki, Y. Tanaka, M.
Tomonaga, N. Yamamoto, and M. Fujii. 2002. Bay 11–7082 inhibits tran-
scription factor NF-?B and induces apoptosis of HTLV-I-infected T-cell
lines and primary adult T-cell leukemia cells. Blood 100:1828–1834.
38. Morita, S., T. Kojima, and T. Kitamura. 2000. Plat-E: an efficient and stable
system for transient packaging of retroviruses. Gene Ther. 7:1063–1066.
39. Nasr, R., E. Chiari, M. El-Sabban, R. Mahieux, Y. Kfoury, M. Abdulhay, V.
Yazbeck, O. Hermine, H. de The, C. Pique, and A. Bazarbachi. 2006. Tax
ubiquitylation and sumoylation control critical cytoplasmic and nuclear steps
of NF-?B activation. Blood 107:4021–4029.
40. Osame, M., K. Usuku, S. Izumo, N. Ijichi, H. Amitani, A. Igata, M. Matsu-
moto, and M. Tara. 1986. HTLV-I associated myelopathy, a new clinical
entity. Lancet 1:1031–1032.
41. Peloponese, J. M., Jr., H. Iha, V. R. Yedavalli, A. Miyazato, Y. Li, K. Haller,
M. Benkirane, and K. T. Jeang. 2004. Ubiquitination of human T-cell leu-
kemia virus type 1 Tax modulates its activity. J. Virol. 78:11686–11695.
42. Petroski, M. D., and R. J. Deshaies. 2005. Function and regulation of cullin-
RING ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 6:9–20.
43. Plans, V., J. Scheper, M. Soler, N. Loukili, Y. Okano, and T. M. Thomson.
2006. The RING finger protein RNF8 recruits Ubc13 for lysine 63-based self
polyubiquitylation. J. Cell. Biochem. 97:572–582.
44. Robek, M. D., and L. Ratner. 1999. Immortalization of CD4?and CD8?T
lymphocytes by human T-cell leukemia virus type 1 Tax mutants expressed in
a functional molecular clone. J. Virol. 73:4856–4865.
45. Rothwarf, D. M., and M. Karin. 1999. The NF-?B activation pathway: a
paradigm in information transfer from membrane to nucleus. Sci. STKE
46. Scheffner, M., J. M. Huibregtse, and P. M. Howley. 1994. Identification of a
human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent
ubiquitination of p53. Proc. Natl. Acad. Sci. USA 91:8797–8801.
47. Senftleben, U., Y. Cao, G. Xiao, F. R. Greten, G. Krahn, G. Bonizzi, Y. Chen, Y.
Hu, A. Fong, S. C. Sun, and M. Karin. 2001. Activation by IKK? of a second,
evolutionary conserved, NF-?B signaling pathway. Science 293:1495–1499.
48. Shembade, N., N. S. Harhaj, D. J. Liebl, and E. W. Harhaj. 2007. Essential
role for TAX1BP1 in the termination of TNF-?-, IL-1- and LPS-mediated
NF-?B and JNK signaling. EMBO J. 26:3910–3922.
49. Smith, M. R., and W. C. Greene. 1990. Identification of HTLV-I Tax trans-
activator mutants exhibiting novel transcriptional phenotypes. Genes Dev.
50. Sun, S. C., and S. Yamaoka. 2005. Activation of NF-?B by HTLV-I and
implications for cell transformation. Oncogene 24:5952–5964.
51. Suzuki, S., P. Singhirunnusorn, A. Mori, S. Yamaoka, I. Kitajima, I. Saiki,
and H. Sakurai. 2007. Constitutive activation of TAK1 by HTLV-1 Tax-
dependent overexpression of TAB2 induces activation of JNK-ATF2 but not
IKK-NF-?B. J. Biol. Chem. 282:25177–25181.
52. Wu, X., and S. C. Sun. 2007. Retroviral oncoprotein Tax deregulates NF-?B
by activating Tak1 and mediating the physical association of Tak1-IKK.
EMBO Rep. 8:510–515.
53. Xiao, G., M. E. Cvijic, A. Fong, E. W. Harhaj, M. T. Uhlik, M. Waterfield,
and S. C. Sun. 2001. Retroviral oncoprotein Tax induces processing of
NF-?B2/p100 in T cells: evidence for the involvement of IKK?. EMBO J.
54. Xiao, G., E. W. Harhaj, and S. C. Sun. 2000. Domain-specific interaction
with the I?B kinase (IKK) regulatory subunit IKK? is an essential step in
Tax-mediated activation of IKK. J. Biol. Chem. 275:34060–34067.
55. Xiao, G., E. W. Harhaj, and S. C. Sun. 2001. NF-?B-inducing kinase regu-
lates the processing of NF-?B2 p100. Mol. Cell 7:401–409.
56. Yamamoto, M., T. Okamoto, K. Takeda, S. Sato, H. Sanjo, S. Uematsu, T.
Saitoh, N. Yamamoto, H. Sakurai, K. J. Ishii, S. Yamaoka, T. Kawai, Y.
Matsuura, O. Takeuchi, and S. Akira. 2006. Key function for the Ubc13 E2
ubiquitin-conjugating enzyme in immune receptor signaling. Nat. Immunol.
57. Yamamoto, M., S. Sato, T. Saitoh, H. Sakurai, S. Uematsu, T. Kawai, K. J.
Ishii, O. Takeuchi, and S. Akira. 2006. Cutting edge: pivotal function of
Ubc13 in thymocyte TCR signaling. J. Immunol. 177:7520–7524.
58. Yamaoka, S., G. Courtois, C. Bessia, S. T. Whiteside, R. Weil, F. Agou, H. E.
Kirk, R. J. Kay, and A. Israel. 1998. Complementation cloning of NEMO, a
component of the I?B kinase complex essential for NF-?? activation. Cell
59. Yao, J., and B. Wigdahl. 2000. Human T cell lymphotropic virus type I
genomic expression and impact on intracellular signaling pathways during
neurodegenerative disease and leukemia. Front. Biosci. 5:D138–D168.
60. Yoshida, M., I. Miyoshi, and Y. Hinuma. 1982. Isolation and characterization
of retrovirus from cell lines of human adult T-cell leukemia and its implica-
tion in the disease. Proc. Natl. Acad. Sci. USA 79:2031–2035.
13742 SHEMBADE ET AL.J. VIROL.