Mutual regulation between deubiquitinase CYLD and retroviral oncoprotein Tax
© Wu et al; licensee BioMed Central Ltd. 2011
Received: 23 June 2011
Accepted: 8 August 2011
Published: 8 August 2011
Oncoprotein Tax, encoded by the human T-cell leukemia virus type 1 (HTLV1), persistently induces NF-κB activation, which contributes to HTLV1-mediated T-cell transformation. Recent studies suggest that the signaling function of Tax requires its ubiquitination, although how the Tax ubiquitination is regulated remains unclear.
We show here that the deubiquitinase CYLD physically interacts with Tax and negatively regulates the ubiquitination of this viral protein. This function of CYLD is associated with inhibition of Tax-mediated activation of IKK although not that of Tak1. Interestingly, CYLD undergoes constitutive phosphorylation in HTLV1-transformed T cells, a mechanism known to inactivate the catalytic activity of CYLD. Consistently, a phospho-mimetic CYLD mutant fails to inhibit Tax ubiquitination.
These findings suggest that CYLD negatively regulates the signaling function of Tax through inhibition of Tax ubiquitination. Conversely, induction of CYLD phosphorylation may serve as a mechanism by which HTLV1 overrides the inhibitory function of CYLD, leading to the persistent activation of NF-κB.
KeywordsCYLD HTLV Tax ubiquitination IKK NF-κB
Human T-cell leukemia virus type 1 (HTLV1) is an oncogenic retrovirus that is etiologically associated with a human acute T-cell malignancy termed adult T-cell leukemia (ATL) [1–3]. HTLV1 genome encodes a 40-kD protein that not only regulates viral gene expression but also induces various cellular genes contributing to HTLV1-mediated T-cell transformation . Tax modulates the activity of different cellular transcription factors, most importantly NF-κB, a family of enhancer-binding proteins regulating cell growth and survival . The activity of NF-κB is normally subject to tight regulation by a cytoplasmic inhibitor, IκB. In response to cellular stimuli, IκB is phosphorylated by a specific IκB kinase (IKK) and targeted for ubiquitination and proteasomal degradation, resulting in nuclear translocation of active NF-κB [6, 7]. Under normal conditions, the activation of IKK and NF-κB occurs transiently, which assures that the expression of NF-κB target genes is induced temporally. However, in HTLV1-infected T cells, Tax persistently stimulates the activity of IKK, leading to constitutive nuclear expression of NF-κB [8–10]. Strong evidence suggests that deregulated NF-κB activation has a central role in HTLV1-mediated T-cell transformation [5, 11, 12].
We and others have previously shown that Tax physically interacts with the IKK complex via the IKK regulatory subunit IKKγ (also called NEMO), and this molecular interaction is critical for Tax-mediated IKK activation [13–15]. More recent work suggests that the signaling function of Tax requires its ubiquitination [16–18]. Although ubiquitination is traditionally viewed as a mechanism that mediates protein degradation in the proteasome, it is now clear that specific types of ubiquitination also facilitate the activation of protein kinases, including IKK . In particular, lysine 63 (K63)-linked polyubiquitin chains may serve as a platform that helps recruit and activate IKK and its activating kinase, Tak1. Like phosphorylation, ubiquitination is a reversible reaction, which is counter-regulated by ubiquitinating enzymes and deubiquitinases (DUBs) . A DUB, CYLD, has been shown to preferentially deconjugate K63-linked ubiquitin chains  and implicated as a negative regulator of IKK/NF-κB signaling. CYLD has constitutive DUB activity, but its activity can be rapidly inactivated via its phosphorylation in response to NF-κB stimuli .
Tax undergos K63 type of ubiquitination, which is critical for activation of NF-κB . However, how the ubiquitination of Tax is regulated remains unclear. In the present study, we have shown that Tax forms a complex with CYLD, in which CYLD strongly inhibits the ubiquitination and signaling function of Tax. Interestingly, in a large panel of HTLV1-transformed T-cell lines, CYLD is constitutively phosphorylated. These findings not only establish CYLD as a negative regulator of Tax ubiquitination but also suggest a mutual regulatory mechanism in which HTLV1 stimulates CYLD phosphorylation and functional inactivation.
Tax physically interacts with CYLD
CYLD inhibits Tax ubiquitination
CYLD inhibits Tax-stimulated activation of IKK but not that of Tak1
CYLD is constitutively phosphorylated in HTLV1-transformed T cells
A phospho-mimetic CYLD mutant failed to inhibit Tax ubiquitination
The data presented in this paper demonstrate a role for CYLD in regulating the ubiquitination of Tax, oncoprotein of the leukemia virus HTLV1 . CYLD is physically assembled into the Tax complex and involved in negative regulation of Tax ubiquitination and signaling function. Furthermore, Tax and CYLD appear to mutually regulate, since CYLD is constitutively phosphorylated in HTLV1-transformed T cells.
Strong evidence suggests that Tax ubiquitination plays a critical role in its signaling function in the NF-κB pathway . Indeed, we have shown that CYLD-mediated Tax deubiquitination is associated with attenuation of IKK activation. Similarly, the ubiquitination and function of Tax are also regulated by another deubiquitinase, USP20 . These findings suggest that ubiquitinated Tax may be targeted by different deubiquitinases, although precisely how the different deubiquitinases regulate Tax during HTLV1 infection remains to be further studied.
Our current study demonstrates that the CYLD-mediated Tax deubiquitination did not affect Tax's ability to activate Tak1. This finding suggests that Tak1 activation is insufficient for Tax-mediated IKK activation, implicating the involvement of different mechanisms in Tax activation of Tak1 and IKK. In further support of this idea, our previous work demonstrates that a Tax mutant, TaxM22, is capable of Tak1 activation despite its defect in IKK activation . One possibility is that Tax not only activates Tak1 but also recruits Tak1 to the IKK complex to mediate IKK activation. Future studies will examine whether Tax ubiquitination is required for recruiting Tak1 to IKK.
The catalytic activity of CYLD is negatively regulated by its phosphorylation . Cellular stimuli induce transient phosphorylation of CYLD, which may contribute to the optimal activation of IKK. A remarkable finding in the present study is that CYLD is constitutively phosphorylated in a large panel of HTLV1-transformed T cells. Since a phospho-mimetic CYLD mutant is defective in inhibiting Tax ubiquitination, the CYLD phosphorylation may contribute to the chronic activation of IKK and NF-κB in HTLV1-transformed T cells. We have previously shown that induction of CYLD phosphorylation by TNF-α or mitogens is mediated by the IKK complex . However, we found that in HEK293 cells, Tax was insufficient for the induction of CYLD phosphorylation (data not shown). This result could be due to the low expression level of IKK components, particularly IKKγ , although the possibility for involvement of additional signaling factors cannot be excluded. Regarding the latter possibility, a recent study suggests that CYLD phosphorylation can also be mediated by the IKK-related kinase IKKε and contributes to IKKε-induced tumorigenesis . Whether IKKε is involved in the CYLD phosphorylation in HTLV1-trnasformed T cells is yet to be investigated. It also remains to be examined whether CYLD phosphorylation also contributes to HTLV1-induced T-cell transformation. Nevertheless, our data suggest that CYLD phosphorylation is a mechanism that mediates constitutive Tax ubiquitination and signaling function in HTLV1-transformed T cells.
The results of this study demonstrate a role for the DUB CYLD in the negative regulation of HTLV1 Tax protein. CYLD inhibits Tax-stimulated IKK activation via deubiquitinating Tax, although the CYLD-mediated Tax deubiquitination does not affect Tax activation of Tak1. Our data further suggest that HTLV1 has developed a mechanism to override the negative-regulatory role of CYLD, and this mechanism involves the induction of CYLD phosphorylation.
Cell lines and transfection
Human embryonic kidney cell line 293, human leukemia T-cell line Jurkat, and HTLV1-transformed human T-cell lines were described previously . The Tax1 cell line is an IL-2-dependent human T-cell line immortalized by HTLV1 Tax in the context of a herpes saimiri vector . For transient transfection, 293 cells were seeded in 6-well plates and transfected using Lipofectamine-2000 (Invitrogen).
Plasmid constructs, antibodies, and other reagents
pCMV4-Tax was kindly provided by Dr. Warner Greene . pcDNA-based expression vectors encoding HA-tagged IKKγ, Ubiquitin, CYLD, a catalytically inactive CYLD mutant (CYLDmut) were as described [14, 22, 41]. CYLD SA is a phosphorylation-deficient mutant harboring alanine substitutions of seven serine residues, and CYLD SE is a phospho-mimetic CYLD mutant harboring serine to glutamic acid substitutions . Ubiquitin mutants K48 and K63 (provided by Dr. Zhijian Chen) carry lysine-to-arginine substitutions in all lysine residues, except K48 and K63, respectively. Ubiquitin mutants K48R and K63R harbor mutations in lysine 48 and 63, respectively. pCMV-HA-Tak1 expression vector and anti-Tak1 antibody were kindly provided by Drs. Kunihiro Matsumoto and Jun Ninomiya-Tsuji . All other reagents were described previously .
Immunoblotting (IB), immunoprecipitation (IP), in vitro kinase assay, and ubiquitination assay
Cell lysates were prepared by lysing the cells in RIPA buffer for IB and IP . For kinase assays, cells were lysed in a kinase lysis buffer supplemented with phosphatase inhibitors and immediately subject to in vitro kinase assays as described previously . For ubiquitination assays, cells were transfected with HA-tagged ubiquitin or its mutants along with other expression vectors. The cells were lysed in RIPA buffer supplemented with 4 mM N-ethylmaleimide (NEM) and immediately bioled for 5 min in the presence of 1% SDS and then diluted 10 times with RIPA buffer. Ubiquitinated Tax was isolated by IP using anti-Tax and detected by IB with anti-HA.
HEK293 cells were transiently transfected with HA-CYLD and Tax and subjected to indirect immunofluorescence assays as described .
List of abbreviations
human T-cell leukemia virus type 1
We thank W.C. Greene for Tax expression vectors, Drs. Jun Niomiya-Tsuji and Kunihiro Matumoto for Tak1 antibody and expression vector, and AIDS Research and Reference Program of NIAID for anti-Tax hybridoma. This study was supported by National Institutes of Health grant AI064639.
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