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. 2011 Aug 8:1:27.
doi: 10.1186/2045-3701-1-27.

Mutual regulation between deubiquitinase CYLD and retroviral oncoprotein Tax

Xuefeng Wu #  1   2 , Minying Zhang #  3   2 , Shao-Cong Sun  2
Affiliations

Mutual regulation between deubiquitinase CYLD and retroviral oncoprotein Tax

Xuefeng Wu et al. Cell Biosci. .

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
Physical interaction between Tax and CYLD. (A) Cell lysates were prepared from the HTLV1-transformed C8166 cell line and subjected to IP using either a control pre-immune serum or anti-CYLD. The precipitated CYLD and its associated Tax were analyzed by IB. (B) HEK293 cells were transfected with HA-CYLD and Tax either in the absence (-) or presence (+) of IKKγ. The cell lysates were subjected to IP using anti-CYLD followed by IB to detect the precipitated CYLD and its associated Tax (top two panels). The cell lysates were also subjected to direct IB to monitor the expression of IKKγ and Tax. (C) HEK293 cells were transfected with HA-CYLD and Tax. The cells were stained with anti-HA (Y-11) and a mouse monoclonal anti-Tax antibody, followed with Texas red-conjugated donkey anti-rabbit Ig and FITC-conjugated donkey anti-mouse Ig. Cells were also counterstained with Hoechst 33258 for nuclear visualization. The expression of CYLD, Tax, Tax-CYLD merge, and nucleus (Hoechst) are shown. Note that the cytoplasmic, but not nuclear, Tax was colocalized with CYLD (Tax-CYLD merge, yellow color).
Figure 2
Figure 2
Tax undergoes K63 ubiquitination, which is inhibited by CYLD. (A) 293 cells were transfected with HA-tagged ubiquitin together with vector control (V) or Tax. Protein lysates were subjected to IP using anti-Tax, and ubiquitin-conjugated Tax and Tax were detected by IB with HRP-anti-HA and anti-Tax, respectively. (B) 293 cells were transfected with Tax together with the indicated ubiquitin mutants. Tax was subject to IP and ubiquitination assays as in A (left panel). Cell lysates were analyzed by IB to monitor ubiquitin expression (right panels). K48R and K63R harbor K/R substitutions at lysines 48 and 63, respectively. K48 and K63 harbor K/R substitutions in all lysines except 48 and 63, respectively. (C) HEK293 cells were transfected with Tax and HA-tagged ubiquitin either the in the absence (-) or the presence of wildtype (WT) CYLD or a catalytically inactive CYLD mutant (Mut). Cell lysates were subjected to Tax ubiquitination (left panel) and IB (right panel) assays. The Tax expression level was comparable between the different lanes (data not shown).
Figure 3
Figure 3
CYLD inhibits Tax-stimulated activation of IKK but not that of Tak1. HEK293 cells were transfected with the indicated expression vectors. Cell lysates were subjected to in vitro kinase assays to detect the activation of endogenous Tak1 (panel 1) and IKK (panel 3). Following autoradiography, the kinase assay membranes were subjected to IB to detect the Tak1 (panel 2) and IKKβ (panel 4) proteins. The expression level of CYLD and Tax was monitored by IB (panels 5 and 6).
Figure 4
Figure 4
Constitutive phosphorylation of CYLD in HTLV1-transformed T cells. Whole-cell lysates were prepared from control Jurkat T cells or the indicated Tax-expressing (Tax+) T cell lines transformed by Tax (Tax1) or HTLV1 (C8166, SLB-1, E55, HUT102) and subjected to IB using anti-CYLD (upper) or anti-IκBα (lower). In lanes 7 and 8, the cell lysates were incubated in vitro with either buffer or calf intestinal phosphatase (CIP) to show that the more slowly migrating protein band is phosphorylated CYLD.
Figure 5
Figure 5
A phospho-mimetic CYLD is defective in inhibiting Tax ubiquitination. HEK293 cells were transfected with Tax and HA-ubiquitin either in the absence (-) or presence of wildtype (WT) CYLD, the catalytically inactive CYLD mutant (Mut), or CYLD mutants harboring serine-to-alanine (SA) or serine-to-glutamic acids (SE) substitutions at its phsophorylation sites. Cell lysates were subjected to Tax ubiquitination (upper) or direct IB to analyze the expression of ubiquitin, CYLD, and Tax (lower three panels).
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