Retroviral oncoprotein Tax induces processing of NF-kappaB2/p100 in T cells: evidence for the involvement of IKKalpha
2001; Springer Nature; Volume: 20; Issue: 23 Linguagem: Inglês
10.1093/emboj/20.23.6805
ISSN1460-2075
AutoresGutian Xiao, Mary Ellen Cvijic, Abraham Fong, Edward W. Harhaj, Mark Uhlik, Michael Waterfield, Shao‐Cong Sun,
Tópico(s)Animal Disease Management and Epidemiology
ResumoArticle3 December 2001free access Retroviral oncoprotein Tax induces processing of NF-κB2/p100 in T cells: evidence for the involvement of IKKα Gutian Xiao Gutian Xiao Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Mary Ellen Cvijic Mary Ellen Cvijic Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Abraham Fong Abraham Fong Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Edward W. Harhaj Edward W. Harhaj Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Mark T. Uhlik Mark T. Uhlik Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Michael Waterfield Michael Waterfield Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Shao-Cong Sun Corresponding Author Shao-Cong Sun Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Gutian Xiao Gutian Xiao Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Mary Ellen Cvijic Mary Ellen Cvijic Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Abraham Fong Abraham Fong Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Edward W. Harhaj Edward W. Harhaj Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Mark T. Uhlik Mark T. Uhlik Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Michael Waterfield Michael Waterfield Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Shao-Cong Sun Corresponding Author Shao-Cong Sun Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA Search for more papers by this author Author Information Gutian Xiao1, Mary Ellen Cvijic1, Abraham Fong1, Edward W. Harhaj1, Mark T. Uhlik1, Michael Waterfield1 and Shao-Cong Sun 1 1Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, 17033 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:6805-6815https://doi.org/10.1093/emboj/20.23.6805 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info IκB kinase (IKK) is a key mediator of NF-κB activation induced by various immunological signals. In T cells and most other cell types, the primary target of IKK is a labile inhibitor of NF-κB, IκBα, which is responsible for the canonical NF-κB activation. Here, we show that in T cells infected with the human T-cell leukemia virus (HTLV), IKKα is targeted to a novel signaling pathway that mediates processing of the nfκb2 precursor protein p100, resulting in active production of the NF-κB subunit, p52. This pathogenic action is mediated by the HTLV-encoded oncoprotein Tax, which appears to act by physically recruiting IKKα to p100, triggering phosphorylation-dependent ubiquitylation and processing of p100. These findings suggest a novel mechanism by which Tax modulates the NF-κB signaling pathway. Introduction The NF-κB family of transcription factors participates in regulation of diverse biological processes, including immune responses, cell growth and apoptosis (Gilmore et al., 1996; Ghosh et al., 1998; Sha, 1998; Barkett and Gilmore, 1999). Mammalian cells express five NF-κB members, RelA, RelB, c-Rel, p50 and p52, which function as various homo- and heterodimers (Siebenlist et al., 1994). The NF-κB factors are normally sequestered in the cytoplasm through physical interaction with ankyrin repeat-containing inhibitors, including IκBα and related proteins (Baldwin, 1996). A well-characterized pathway leading to NF-κB activation is through phosphorylation and subsequent degradation of IκBα (Brockman et al., 1995; Brown et al., 1995). This canonical NF-κB signaling pathway depends on a multisubunit IκB kinase (IKK), which responds to various stimuli, such as the inflammatory cytokine tumor necrosis factor α (TNF-α), the mitogen phorbol 12-myristate 13-acetate (PMA) and certain viral proteins (Sun and Ballard, 1999; Karin and Ben-Neriah, 2000; Hiscott et al., 2001). The IKK is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ (also named NEMO, IKKAP1 or FIP-3) (Karin and Ben-Neriah, 2000). Recent gene knockout studies suggest that the two catalytic subunits of IKK have distinct physiological functions. While IKKβ is essential for signal-induced IκBα phosphorylation, IKKα is largely dispensable for this function (Hu et al., 1999; Li et al., 1999a,b,d; Tanaka et al., 1999). IKKα appears to regulate keratinocyte differentiation in a NF-κB- independent manner (Hu et al., 2001). Another level of NF-κB regulation is via processing of the NF-κB1 and NF-κB2 precursor proteins p105 and p100, a proteasome-catalyzed event required to generate p50 and p52, respectively (Fan and Maniatis, 1991; Siebenlist et al., 1994). These precursor proteins contain ankyrin repeats at their C-terminal portions and function as IκB-like NF-κB inhibitors (Rice et al., 1992; Mercurio et al., 1993). The processing of p105 and p100 not only generates mature p50 and p52 but also results in liberation of the sequestered NF-κB members. By and large, the processing of p105 occurs constitutively and seems to be mediated primarily by a cotranslational mechanism (Lin et al., 1998a, 2000). Although p105 serves as a target of IKK, the IKK-mediated p105 phosphorylation may regulate signal-induced degradation, rather than limited processing, of p105 (Belich et al., 1999; Heissmeyer et al., 1999). However, inducible processing of p105 may occur under certain conditions (Orian et al., 2000). In contrast to p105, the cotranslational processing of p100 is extremely poor (Heusch et al., 1999), and p100 does not undergo inducible processing or degradation in response to various cellular stimuli (Sun et al., 1994). The poor basal processing of p100 seems to be due to the presence of a C-terminal processing-inhibitory domain (PID) (Xiao et al., 2001). Thus, in most cell types, p100 is expressed largely as its unprocessed form, while a large proportion of p105 is processed to p50. Consistently, p50 forms prototypical NF-κB heterodimers, but p52 is only involved in specific functions of NF-κB such as B-cell growth and formation of germinal centers in peripheral lymphoid organs (Caamano et al., 1998; Franzoso et al., 1998). Although the cellular signals stimulating p100 processing remain unknown, the NF-κB-inducing kinase (NIK) has been shown to play a key regulatory role in this proteolysis event (Xiao et al., 2001). Expression of NIK, but not of other MAP kinase kinase kinases (MAP3Ks), in mammalian cells induces p100 processing (Xiao et al., 2001). Consistently, nik gene mutation in alymphoplasia (aly) mice leads to a block of p100 processing in vivo (Xiao et al., 2001). The NIK-induced p100 processing involves site-specific phosphorylation and subsequent ubiquitylation of p100, although it is unclear whether NIK or a NIK-associated kinase catalyzes the p100 phosphorylation (Xiao et al., 2001). A recent study indicates that IKKα may be involved in NIK-induced p100 processing (Senftleben et al., 2001). NIK does not induce p100 processing in mouse embryonic fibroblasts (MEF) lacking IKKα, and this defect can be rescued by transfection of IKKα. However, since many cellular stimuli capable of activation of both IKKα and IKKβ fail to induce p100 processing, it is clear that a novel mechanism is involved in the NIK/p100 pathway. It is likely that NIK-induced p100 processing may involve additional mechanisms other than activation of IKKα. The tight control of p52 generation may be important for proper regulation of NF-κB function in cell growth and survival. Indeed, emerging evidence suggests that deregulated production of p52 may cause abnormal lymphocyte proliferation and transformation. Mice overexpressing p52, in the absence of its precursor p100, develop gastric and lymphoid hyperplasia (Ishikawa et al., 1997). In humans, the nfκb2 gene is frequently involved in chromosomal translocations associated with various lymphomas (Rayet and Gelinas, 1999). In all cases studied, the rearranged nfκb2 genes encode p100 mutants lacking their C-terminal region (Rayet and Gelinas, 1999), which contains the PID, thus rendering them capable of constitutive processing (Xiao et al., 2001). Interestingly, overproduction of p52 is also associated with T-cell transformation induced by the human T-cell leukemia virus type 1 (HTLV-I) (Lanoix et al., 1994). HTLV-I is an oncogenic retrovirus etiologically associated with the development of an acute T-cell malignancy, adult T-cell leukemia (ATL) (Poiesz et al., 1980; Yoshida et al., 1982). HTLV-I transforms T cells via its regulatory protein Tax, which acts by inducing aberrant expression of a large array of cellular genes involved in T-cell growth and survival (Ressler et al., 1996). Tax induces many of these genes through activation of the transcription factor NF-κB (Sun and Ballard, 1999). Recent studies suggest that Tax physically associates with IKK (Chu et al., 1998) and stimulates the catalytic activity of this cellular kinase (Geleziunas et al., 1998; Uhlik et al., 1998; Yin et al., 1998; reviewed by Sun and Ballard, 1999). This virus-specific effect is dependent on IKKγ (Yamaoka et al., 1998; Harhaj et al., 2000), which serves as an adaptor for recruiting Tax to the IKK catalytic subunits (Chu et al., 1999; Harhaj and Sun, 1999; Jin et al., 1999; Xiao et al., 2000). Tax-induced IKK activation is responsible for the persistent phosphorylation of IκBα and nuclear expression of NF-κB in HTLV-infected T cells (Sun and Ballard, 1999). Since IKK activation by cellular stimuli does not enhance p100 processing, it has remained unclear how HTLV-I induces active production of p52. In this study, we have demonstrated that the Tax protein functions as a potent inducer of p100 processing. Tax interacts specifically with p100 via two short helices, and this molecular interaction is essential for Tax induction of p100 processing. Interestingly, the Tax-induced p100 processing does not require NIK but involves the non-canonical IKK component IKKα, which phosphorylates specific serines at the C-terminal region of p100. An important mechanism of Tax action in this virus-specific pathway is to recruit IKKα to p100. These studies provide an example of how a retroviral oncoprotein modifies the function of a cellular protein kinase and targets it to a pathogenic pathway. Results Active processing of p100 in HTLV-I infected human T cells To investigate the mechanism underlying the aberrant expression of p52 in HTLV-infected T cells, we examined whether HTLV infection induces processing of p100. The level of p100 and p52 was analyzed in a large panel of HTLV-transformed T-cell lines, established either from ATL patients or by in vitro HTLV infection of human primary T cells (Uhlik et al., 1998). In two HTLV-negative control T-cell lines (Jurkat and Sup T1), little p52 could be detected (Figure 1A); in contrast, a remark ably high level of p52 was detected in each of the HTLV-transformed T-cell lines (Figure 1A and see Supplementary figure 1 available at The EMBO Journal Online). The amount of p100 was relatively high in the HTLV-positive cells; this was likely due to nfκb2 gene induction by the activated NF-κB in these cells (Liptay et al., 1994; Sun et al., 1994). Indeed, activation of NF-κB in the control Jurkat cells by mitogen treatment also led to heightened expression of p100 (lane 11). However, consistent with the inability of p100 to respond to cellular activation signals (Sun et al., 1994), the level of p52 in the mitogen-stimulated cells was still extremely low (lane 11); a faint p52 band could be detected only after prolonged exposure of the immunoblotting films (data not shown). A similar result was obtained with TNF-α-stimulated T cells (data not shown). Thus, the induction of p100 processing in HTLV-transformed T cells appeared to be mediated specifically by HTLV. This idea was confirmed in freshly isolated human T cells. When these cells were activated by the polyclonal T-cell activator phytohemagglutinin (PHA), only a little p52 could be detected, although these cells expressed an abundant level of p100 (Figure 1B, lane 1). In contrast, infection of the cells with HTLV led to potent p52 production (lane 2). A parallel control immunoblotting assay showed that the level of the RelA subunit of NF-κB was comparable in both the normal and HTLV-infected T cells. These results clearly demonstrate that HTLV infection induces abnormal processing of p100. Figure 1.Active processing of p100 associated with HTLV-I infection. (A) Constitutive processing of p100 in HTLV-transformed T cell lines. Whole-cell extracts (20 μg), isolated from the control or HTLV-infected T-cell lines, were subjected to IB using anti-p100 (upper panel) or anti-actin (lower panel) antibodies. In lane 11, the cells were stimulated with the mitogen PMA (10 ng/ml) and ionomycin (1 μM) for 8 h before extract preparation. (B) Processing of p100 induced by HTLV-I infection of human primary T cells. Cell lysates were isolated from PHA-stimulated or HTLV-infected human primary T cells and subjected to IB using the antibodies indicated. (C) Pulse–chase labeling. Jurkat cells (stimulated for 2 h with PMA and ionomycin, P/I) or HTLV-infected C8166 cells were pulse-labeled for 45 min followed by chase for the time periods indicated. In lane 11, the cells were chased in the presence of a proteasome inhibitor, MG132 (50 μM). p100 and its processing product p52 are indicated. Two non-specific bands are indicated by ns. (D) Densitometry quantitation of the radiolabeled p100 and p52 bands presented in (C). Download figure Download PowerPoint We then performed a pulse–chase labeling study to examine whether HTLV-induced p100 processing occurs at the post-translational or cotranslational levels. As shown in Figure 1C and D, in HTLV-infected T cells, p52 was actively generated from the pulse-labeled precursor protein p100, which was sensitive to a proteasome inhibitor, MG132 (lane 11). The precursor–product relationship was most evident within the first 3 h of chase (Figure 1D, right panel), but the processing rate was gradually retarded after longer periods of chase. Although the mechanism mediating such a pattern of processing remains unclear, it may involve feedback inhibition by the processed product (p52), a mechanism proposed for the regulation of p105 processing (Harhaj et al., 1996). During the late time points of chase, the level of p100 in Jurkat cells was also decreasing, but it was not associated with significant generation of p52 (Figure 1D, left panel). This slow loss of p100 was likely due to protein decay, although it might also result from mitogen-stimulated degradation (the Jurkat cells were stimulated with mitogens). Nevertheless, generation of p52 was clearly a specific event for the HTLV-infected cells. These results suggest the involvement of a post-translational mechanism in HTLV-induced p100 processing. It remains to be determined whether Tax also enhances the cotranslational processing of p100, a mechanism known to mediate the basal processing of p100 (Heusch et al., 1999). Induction of p100 processing is mediated by the Tax oncoprotein Since Tax serves as the key mediator of HTLV-induced T-cell transformation (Ressler et al., 1996), we investigated whether this viral protein was responsible for the induction of p100 processing. Jurkat T cells were infected with retroviral expression vectors encoding either Tax or the green fluorescent protein (GFP), followed by analysis of the processing of endogenous p100. Expression of Tax led to active production of p52 (Figure 2A, lane 5), a result reminiscent of that obtained with HTLV-infected T cells (see Figure 1). The aberrant p100 processing was also detected in a Tax-transformed T-cell line, Tax1 (lane 6), which was generated by delivering the Tax cDNA into human cord blood T cells (Grassmann et al., 1992). In contrast, GFP expression in Jurkat cells did not alter the fate of p100 (lane 3), as compared with uninfected cells (lane 1). Moreover, mitogen stimulation of both uninfected and GFP-infected cells failed to induce p52 production (lanes 2 and 4). Thus, Tax appeared to be an inducer of p100 processing. We further examined this possibility by transient transfection studies. When expressed in Jurkat cells, the exogenous p100 remained largely unprocessed (Figure 2B, upper panel, lane 1), and this state was not altered by mitogen stimulation (lane 2). However, the p100 was processed efficiently when it was coexpressed with Tax (lane 3). A similar result was obtained with non-lymphoid 293 cells (Figure 2C). These results further demonstrate that the productive processing of p100 associated with HTLV infection results from a specific action of Tax. Figure 2.Induction of p100 processing by Tax. (A) Jurkat cells were either not infected or infected with retroviral vectors encoding GFP or Tax. The cells were either not treated (–) or stimulated for 8 h with PMA plus ionomycin (+) followed by extract preparation and IB analysis using anti-p100 (upper panel) or anti-actin (lower panel). In lane 6, IB was performed using an extract isolated from a Tax-transformed T-cell line, Tax1. Non-specific bands are indicated by ns. (B) Jurkat cells (5 × 106) were transfected with p100 (1 μg) together with either an empty vector or Tax (1 μg) followed by mitogen treatment as indicated. Exogenous p100/p52 and Tax were analyzed by IB using anti-p100 (upper panel) and anti-Tax (lower panel). (C) Induction of p100 processing by Tax in 293 cells. 293 cells were transfected with p100 (0.5 μg) together with either an empty vector or Tax (1 μg) followed by IB assays. Download figure Download PowerPoint IKKγ, but not NIK, is essential for Tax-induced p100 processing We have recently shown that NIK is a kinase that specifically induces p100 processing, although the cellular signals triggering this cellular pathway remain unknown (Xiao et al., 2001). We investigated the role of NIK in Tax-induced p100 processing by dominant-negative inhibition assays. The C-terminal portion of NIK (NIK-C) is known to serve as a potent inhibitor of the wild-type NIK (Lin et al., 1998b). Consistently, we found that overexpression of NIK-C efficiently blocked NIK-induced p100 processing (Figure 3A, lanes 5–7). However, NIK-C had no effect on Tax-induced p100 processing (lanes 2–4). A similar result was obtained with a catalytically inactive NIK (lanes 8–13). This result indicates strongly that NIK is not involved in Tax induction of p100 processing. Parallel reporter gene assays showed that the NIK mutants partially inhibited Tax-induced κB enhancer activity and more efficiently inhibited the NIK-induced κB activation (Figure 3B). Since the Tax-induced p100 processing was even slightly enhanced by the NIK mutants, the inhibitory effect of NIK mutants on Tax activation of κB may not be linked to the p100 processing. Figure 3.Effect of dominant-negative NIK mutants on Tax- and NIK-induced p100 processing. (A) 293 cells were transfected with Tax (1 μg) or HA-tagged NIK (0.8 μg) together with the indicated amounts of HA-tagged NIK(650–947) (NIK-C) or NIK(KK429–430AA) (NIK-KA). All the cells were also transfected with p100 (0.5 μg). The processing of p100 and expression of HA-tagged NIK proteins were analyzed by IB using anti-p100 (upper panel) and anti-HA (lower panel), respectively. Wild-type NIK and NIK-KA comigrate in the gel. (B) 293 cells were transfected as in (A) except that p100 was replaced with κB–TATA–luciferase and a control Rennila luciferase reporter driven by the constitutive thymidine kinase promoter. Dual luciferase assays were performed as described in Materials and methods. The κB-specific luciferase activity is presented as fold induction relative to the basal level measured in cells transfected with the pcDNA empty vector. The values shown are representative of three independent experiments. Download figure Download PowerPoint To further assess the mechanism by which Tax induces p100 processing, we analyzed the effect of two well-studied Tax mutants, M22 and M47 (Smith and Greene, 1990), on this specific function. M22 is defective in IKK activation due to its inability to bind IKKγ, while M47 retains this specific activity (Chu et al., 1999; Harhaj and Sun, 1999; Jin et al., 1999). Interestingly, M47, but not M22, induced the processing of p100 (Figure 4A, lanes 3 and 4). This finding indicated the involvement of the IKK signaling pathway in Tax-induced p100 processing. We tested this idea further using an IKKγ-deficient Jurkat T-cell mutant, JM4.5.2, which is completely defective in NF-κB activation by both cellular stimuli and Tax (Harhaj et al., 2000). Tax was delivered to the wild-type or mutant Jurkat cells via retroviral infection. As expected, Tax induced p100 processing in the parental Jurkat cells (Figure 4B, lane 2). However, this proteolytic event did not occur in the IKKγ−/− cells (lane 3). This defect was completely rescued when the JM4.5.2 cells were reconstituted with exogenous IKKγ (lane 4). Expression of exogenous IKKγ alone or together with mitogens did not induce the processing of endogenous p100 (lanes 7 and 8), although IKKγ reconstitution rescued the induction of p100 de novo synthesis by the mitogens (lane 8). Figure 4.Requirement of IKK in Tax-induced p100 processing. (A) Induction of p100 processing by Tax and its mutants. 293 cells were transfected with an empty vector or the indicated Tax constructs followed by analyzing the p100 processing and expression of Tax by IB. (B) Parental Jurkat (Wt), an IKKγ-deficient (γ−/−) Jurkat derivative JM4.5.2 or an IKKγ-reconstituted JM4.5.2 [γ−/−(IKKγ)] (Rivera-Walsh et al., 2000) was infected with retroviral vectors encoding GFP or Tax, followed by IB analysis of endogenous p100/p52 (upper panel) and infected Tax (lower panel) (lanes 1–4). In lanes 5–8, the uninfected cells were either not treated (–) or stimulated for 8 h with PMA plus ionomycin (+), followed by IB analysis of endogenous p100/p52 (upper panel) and actin (lower panel). (C) Transient transfection was performed with the parental and IKKγ-deficient Jurkat cells and the expression vectors indicated (1 μg for p100 and Tax, 0.2 μg for IKKγ, 0.8 μg for NIK; V stands for pcDNA vector). Processing of exogenous p100 was analyzed by IB. Download figure Download PowerPoint To confirm that the lack of p52 production in the IKKγ-deficient T cells was not due to the low level of p100, exogenous p100 was transfected to the parental and IKKγ-deficient Jurkat T cells either alone or together with Tax (Figure 4C). Tax-induced processing of the exogenous p100 occurred in wild-type (lane 2) but not IKKγ-deficient cells (lane 3), unless IKKγ was transfected (lane 4). Thus, IKKγ is an essential factor involved in Tax-induced p100 processing. Interestingly, in a parallel experiment, we found that NIK induced p100 processing in both the wild-type and IKKγ-deficient T cells (Figure 4C, lanes 7 and 10). This finding provides another piece of evidence indicating that Tax-induced p100 processing is not mediated through NIK. Tax-induced p100 processing requires Tax–p100 physical interaction An important question to consider is why other IKK stimulators could not induce p100 processing. One potential answer is that Tax not only activates IKK but may also functionally modify this cellular kinase in the induction of p100 processing. In this regard, Tax is known to interact physically with p100 (Béraud et al., 1994). Although the functional significance of this molecular interaction remains unclear, it is interesting to note that the M22 mutant of Tax, incapable of inducing p100 processing (Figure 4A), is inactive in p100 binding (Béraud et al., 1994). To assess the role of the Tax–p100 interaction in regulating p100 processing, we mapped the domain within p100 involved in Tax interaction. Interestingly, a region of p100 containing two short helices was found to be important for Tax binding (Figure 5A and B). Prior structural studies demonstrated that these two helices (αA and αB) (Figure 5A) are exposed on the surface of the protein but are not involved in DNA binding, dimerization or general folding of p52 (Cramer et al., 1997). It has been proposed that this region of p100 may interact with certain regulatory proteins (Cramer et al., 1997). Both αA and αB appeared to be involved in the binding of p100 to Tax. Deletion of αA partially inhibited the p100–Tax interaction (Figure 5B, upper panel, lane 3), while deletion of both helices largely abolished the binding (lane 4). Similarly, disruption of the α helices by proline substitutions also prevented the binding of p100 to Tax (lane 6). On the other hand, none of these structural alterations affected the dimerization function of p100 (Figure 5C), which was in agreement with the previous structural studies. More importantly, the p100 mutants defective in Tax binding failed to respond to Tax-induced processing (Figure 5B, middle panel, lanes 4 and 6). These results indicate that Tax associates physically with p100 via two α helices, and this interaction is essential for Tax induction of p100 processing. Figure 5.Interaction of Tax with p100 via two α helices, which is correlated with Tax-induced p100 processing. (A) Scheme of p100 indicating the two α helices, αA and αB, previously identified by X-ray crystallography (Cramer et al., 1997). (B) 293 cells were transfected with Tax together with the indicated p100 constructs. The p100 proteins were isolated from cell lysates by IP followed by detection of the coprecipitated Tax by IB (upper panel). The cell lysates were also subjected to IB to monitor the p100 processing (middle panel) and Tax expression (lower panel). (C) Dimerization of p100 mutants with the Rel homology domain of RelA, RelA(1–312). 293 cells were transfected with RelA(1–312) together with the p100 constructs, followed by coIP assays. Download figure Download PowerPoint Tax recruits IKKα to p100 Since Tax interacts with both IKK (via IKKγ) (Chu et al., 1999; Harhaj and Sun, 1999; Jin et al., 1999) and p100, this raised the intriguing possibility that Tax specifically targets the IKK complex to p100, triggering phosphorylation-dependent p100 processing. We examined whether IKK is in the same complex with p100 in control Jurkat cells and HTLV-infected cells by coimmunoprecipitation (coIP). No significant binding between p100 and IKKα was detected in either untreated (data not shown) or mitogen-stimulated Jurkat T cells (Figure 6A, lane 2). Interestingly, a stable p100–IKKα association was readily demonstrated in the HTLV-infected C8166 cells (lane 4) as well as a number of other HTLV-infected T-cell lines (data not shown). The levels of IKKα and p100 proteins were similar in the mitogen-treated Jurkat cells and the C8166 cells (lanes 5 and 6). Interestingly, a parallel coIP assay revealed that IKKβ was not associated with the p100 complex (Figure 6B, upper panel, lane 4). This biochemical defect was not due to the inefficiency of the IKKβ antibody in IP, since it efficiently pulled down Tax (lower panel, lane 4). This finding suggests that although both IKKα and IKKβ form complexes with Tax (via IKKγ), only IKKα is present in the p100–Tax complex. Figure 6.Induction of p100–IKKα binding by Tax. (A) Stable association of p100 with IKKα in HTLV-infected T cells. Cell extracts were prepared from mitogen-stimulated Jurkat cells or the HTLV-infected C8166 cells and subjected to IP using either a preimmune serum (PI) or anti-p100 antibody, and the coprecipitated IKKα was detected by IB (lanes 1–4). The extracts were also analyzed directly by IB to detect the expression levels of IKKα, p100, Tax and actin (lanes 5 and 6). (B) IKKα, but not IKKβ, is in the p100 complex in HTLV-infected T cells. The C8166 cell lysates were subjected to IP using the indicated PI and immune sera. The p100 (upper panel) and Tax (lower panel) proteins in the immune complexes were analyzed by IB. (C) Binding of transfected p100 to endogenous IKKα in Tax-express
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