IKKγ Mediates the Interaction of Cellular IκB Kinases with the Tax Transforming Protein of Human T Cell Leukemia Virus Type 1*

The Tax oncoprotein of human T cell leukemia virus type 1 constitutively activates transcription factor NF-κB by a mechanism involving Tax-induced phosphorylation of IκBα, a labile cytoplasmic inhibitor of NF-κB. To trigger this signaling cascade, Tax associates stably with and persistently activates a cellular IκB kinase (IKK) containing both catalytic (IKKα and IKKβ) and noncatalytic (IKKγ) subunits. We now demonstrate that IKKγ enables Tax to dock with the IKKβ catalytic subunit, resulting in chronic IκB kinase activation. Mutations in either IKKγ or Tax that prevent formation of these higher order Tax·IKK complexes also interfere with the ability of Tax to induce IKKβ catalytic function in vivo. Deletion mapping studies indicate that amino acids 1–100 of IKKγ are required for this Tax targeting function. Together, these findings identify IKKγ as an adaptor protein that directs the stable formation of pathologic Tax·IKK complexes in virally infected T cells.


The Tax oncoprotein of human T cell leukemia virus type 1 constitutively activates transcription factor NF-B by a mechanism involving Tax-induced phosphorylation of IB␣, a labile cytoplasmic inhibitor of NF-B.
To trigger this signaling cascade, Tax associates stably with and persistently activates a cellular IB kinase (IKK) containing both catalytic (IKK␣ and IKK␤) and noncatalytic (IKK␥) subunits. We now demonstrate that IKK␥ enables Tax to dock with the IKK␤ catalytic subunit, resulting in chronic IB kinase activation. Mutations in either IKK␥ or Tax that prevent formation of these higher order Tax⅐IKK complexes also interfere with the ability of Tax to induce IKK␤ catalytic function in vivo. Deletion mapping studies indicate that amino acids 1-100 of IKK␥ are required for this Tax targeting function. Together, these findings identify IKK␥ as an adaptor protein that directs the stable formation of pathologic Tax⅐IKK complexes in virally infected T cells.
During an adaptive immune response, antigen-stimulated CD4 ϩ T lymphocytes become committed to an activation program that triggers a transient phase of clonal expansion (1). In contrast, infection with human T cell leukemia virus type 1 (HTLV-1) 1 can lead to the loss of cell cycle control and development of an aggressive malignancy called adult T cell leukemia (2). The Tax oncoprotein encoded by HTLV-1 stimulates the constitutive nuclear expression of transcription factor NF-B, which regulates antigen-directed T cell proliferation (3,4).
Studies with Tax-transgenic mice suggest that this viral/host interaction is required to maintain the transformed phenotype of HTLV-1-infected cells (5).
In quiescent T cells, the activity of NF-B is controlled from the cytoplasmic compartment by virtue of its signal-dependent interaction with inhibitors, including IB␣ (6). Recent studies have identified two cytokine-inducible IB kinases (IKKs), termed IKK␣ and IKK␤, that target IB␣ for degradation via phosphorylation at Ser-32 and Ser-36 (7). These two kinases form heterodimers and function as catalytic subunits within a 700 -900-kDa multicomponent complex (8). Whereas IKK␣ and IKK␤ are activated transiently in cells treated with the cytokine tumor necrosis factor-␣ (TNF) (8 -10), Tax induces their constitutive expression in HTLV-1-infected T cells (11,12). We have recently found that Tax-induced activation of both IKK and NF-B requires the formation of Tax⅐IKK complexes (12). However, the precise mechanism of Tax action on IKKs remains unclear.
Here we provide several lines of experimental evidence indicating that Tax-directed IKK activation is mediated by IKK␥ (also called NEMO, IKKAP1, or FIP-3), a recently identified subunit of TNF-responsive IKKs whose precise signaling function is unknown (13)(14)(15)(16). First, interference with IKK␥ expression in T cell transfectants inhibits Tax-mediated activation of NF-B. Second, IKK␥ and Tax interact stably in the context of a high molecular mass IB kinase derived from HTLV-1-infected T cells. Third, overexpression of IKK␥ in vivo is sufficient to target Tax specifically to ectopic IKK␤, whereas deletion of the N-terminal region of IKK␥ eliminates this targeting function. The finding that IKK␥ enables Tax to dock with cellular IB kinases highlights an important missing link in the mechanism by which this oncoprotein activates the constitutive expression of NF-B in HTLV-1-infected T cells.

Constitutive Activation of NF-B by Tax Involves IKK␥ but
Not RIP-In prior studies, we established that Tax binds to and persistently activates a TNF-responsive IB kinase containing two catalytic subunits termed IKK␣ and IKK␤ (12). To determine how Tax interfaces with cellular IKKs, we first examined whether the Tax⅐IKK signaling axis involves the death domain kinase RIP, which is essential for TNF-induced activation of NF-B (20,28). For these studies, Jurkat human T cells containing a RIP null mutation (20) were cotransfected with a Tax expression vector (Tax-WT) and a CAT reporter plasmid containing two B enhancers (B-TATA-CAT). Parallel experiments were conducted with expression vectors containing point mutations that selectively disrupt the ability of Tax to access either the CREB/ATF (Tax-M47) or the NF-B/Rel (Tax-M22) transcription factor pathway (17). As shown in Fig. 1A, Tax-WT potently stimulated NF-B-directed transcription in both parental and RIP-deficient Jurkat T cells. Similar results were obtained with Tax-M47 but not Tax-M22, consistent with their differing capacities to activate TNF-responsive IKKs (12). These in vivo functional data clearly show that RIP is dispensable for Tax-induced activation of NF-B.
RIP interacts specifically with IKK␥, an integral subunit of TNF-responsive IKKs (13)(14)(15)(16). In this regard, Yamaoka et al. (13) have reported experiments with IKK␥-deficient rat fibroblasts, suggesting a requirement for this subunit in coupling Tax to NF-B, whereas others (29) have identified an IKK␥deficient pre-B cell line that is fully responsive to Tax. To determine whether IKK␥ couples Tax to NF-B in a more physiologically relevant setting, Jurkat T cells were cotransfected with Tax-WT, B-TATA-CAT, and graded amounts of a vector that directs the synthesis of antisense IKK␥ RNA (AS-IKK␥). As shown in Fig. 1B, AS-IKK␥ inhibited Tax-induced transcription directed from the NF-B-responsive reporter in a dose-dependent fashion. In contrast, interference with IKK␥ protein expression failed to affect Tax-induced transcription from the HTLV-1 5Ј long terminal repeat, which is activated by an NF-B-independent mechanism (30). These functional studies demonstrate that IKK␥ is required for the induction of NF-B by Tax in the context of human T lymphocytes, the in vivo target for HTLV-1.
Tax Interacts Stably with TNF-responsive IKKs Containing IKK␥-IKK␥ assembles with TNF-responsive IKKs primarily via its interaction with IKK␤ (13)(14)(15). This catalytic subunit also associates with Tax in HTLV-1-infected T cells (12). To determine whether IKK␥ is a core component of Tax-responsive IKKs, we first performed in vitro kinase assays using cytoplasmic extracts from SLB-1 and C8166 T cells. Whereas SLB-1 cells produce replication-competent virions (21), C8166 cells harbor a defective provirus that selectively expresses Tax (22). In these biochemical experiments, Tax, IKK␣, IKK␤, and IKK␥ were isolated by immunoprecipitation and assayed for IKK catalytic activity using a GST-IB␣ fusion protein as substrate. As shown in Fig. 2A (lanes 2-4, top panel), GST-IB␣ phosphorylating activity was readily detected in Tax and IKK␣⅐IKK␤ immunoprecipitates derived from SLB-1 cells. A significant amount of IKK activity was also detected in IKK␥ immunoprecipitates (lane 5). Similar results were obtained with C8166 cells (Fig. 2A, bottom panel). This subunit compositional analysis establishes that IKK␥ is associated with a constitutively active IB kinase in both HTLV-1-infected and Tax-expressing T cells.
The TNF-responsive form of IKK that contains IKK␥ corresponds to a 700 -900-kDa multisubunit complex (13,14). To explore the size distribution of Tax-associated IKKs, cytosolic proteins from HTLV-1-infected SLB-1 cells were fractionated by gel filtration and the resultant eluates were subjected to immunoprecipitation with anti-Tax antibodies. Consistent with the size of TNF-responsive IKKs, the majority of constitutively active IKKs associated with Tax in SLB-1 cells were detected in fractions corresponding to a molecular mass exceeding 700 kDa (Fig. 2B). To determine whether IKK␥ was also present in these Tax⅐IKK complexes, SLB-1 fractions containing peak kinase activity were subjected to immunoprecipitation with IKK␥-specific antibodies and assayed for the presence of either IKK activity or Tax protein. As shown in Fig. 2C, IB kinase activity and Tax were readily detected in these IKK␥ immunoprecipitates (lane 3, top and bottom panels). These biochemical data indicate that Tax associates with IKK␥ in the context of a high molecular mass IB kinase in HTLV-1-infected T cells.
To address the stability of these higher order Tax⅐IKK␥ complexes, IKK␥ was immunopurified from SLB-1 fractions containing peak kinase activity (Ͼ700 kDa) and washed at high stringency with escalating concentrations of NaCl and urea. We then monitored the dissociation of IKK␣⅐IKK␤ and Tax from these IKK␥ immunocomplexes using in vitro kinase and immunoblotting assays, respectively. As shown in Fig. 2D (bottom panel), high concentrations of either dissociation agent failed to release Tax from IKK␥. Interactions between IKK␥ and the IKK␣⅐IKK␤ catalytic subunits were also highly resistant to release, as inferred from our ability to detect significant levels of IKK␥-associated IB kinase activity under identical washing conditions (Fig. 2D, top panel). These results confirm that Tax, IKK␥, and the catalytic subunits of IKK interact with high affinity, further underscoring the specificity and pathologic relevance of this viral/host interaction in HTLV-1-infected T cells.
IKK␥ Mediates the Functional Interaction between Tax and IKK␤-Tax activates TNF-responsive IKKs primarily via its stimulatory effects on IKK␤ (11), which interacts directly with IKK␥ (13)(14)(15). To determine whether IKK␥ directs the assembly of Tax⅐IKK complexes, mammalian 293T cells were transfected with expression vectors for FLAG-tagged IKK␤, IKK␥, and Tax. Cytoplasmic extracts were then prepared and subjected to immunoprecipitation with either monoclonal anti-FLAG antibodies (Fig. 3A, top and middle panels) or Taxspecific antibodies (Fig. 3A, bottom panel). When IKK␤ immunocomplexes were probed on immunoblots for the presence of Tax, we found that IKK␤ interacted weakly with Tax-WT, Tax-M22, and Tax-M47 in IKK␥-deficient cells (Fig. 3A,  lanes 2, 5, and 8, top panel). In contrast, significant amounts of both Tax-WT and Tax-M47 were associated with IKK␤ in cells coexpressing IKK␥ (lanes 3 and 9). Under identical transfection conditions, Tax-M22 failed to interact appreciably with IKK␤ in the presence of ectopic IKK␥ (lane 6). This divergent result with Tax-M22 could not be attributed to inefficient ectopic expression, because comparable amounts of IKK␥ and Tax protein were detected in each triple transfection (Fig. 3A, lanes  3, 6, and 9, middle and bottom panels). Coupled with our prior observation that Tax-M22 is defective for binding to endogenous IKKs (12), these data strongly suggest that IKK␥ confers IKK␤ targeting specificity to Tax.
To explore the functional consequences of these higher order interactions, expression vectors for IKK␤, IKK␥, and Tax were introduced into S107 plasmacytoma cell line. Importantly, S107 cells harbor a genetic defect that impairs NF-B expression (23), thus providing a cellular background with minimal IB kinase activity. Following transfection, ectopic IKK␤ was immunopurified from S107 cytoplasmic extracts and monitored for catalytic activity using an in vitro kinase assay. As shown in Fig. 3B, all three of the Tax constructs failed to stimulate IKK␤ kinase activity in the absence of ectopic IKK␥ (lanes 2-4), whereas overexpression of IKK␥ in cells harboring wild type Tax and Tax-M47 potently induced IKK␤ (lanes 6 and 8). In contrast, Tax-M22 was unable to activate IKK␤ in the presence of IKK␥ (lane 7), consistent with its defect in endogenous IKK binding (12). These functional data correlate precisely with our biochemical results demonstrating that IKK␥ directs the formation of Tax-M47⅐IKK␤ but not Tax-M22⅐IKK␤ complexes in mammalian 293T cell transfectants (Fig. 2B).
The N Terminus of IKK␥ Is Required for Tax Targeting to IKK␤-Primary sequence analyses indicate that IKK␥ contains a C-terminal leucine zipper domain, a central coiled-coil domain, and an N-terminal domain with no apparent secondary structural features (13)(14)(15)(16). To define the sequences in IKK␥ that mediate its Tax adaptor function, Tax and FLAG-tagged IKK␤ were transiently expressed in 293T cells along with a panel of Myc epitope-tagged deletion mutants of IKK␥ (Fig.  4A). Cytoplasmic extracts were prepared from transfected cells and subjected to immunoprecipitation with antibodies specific for each ectopic protein. The resultant immunocomplexes were then probed for the presence of Tax and IKK␥ protein on immunoblots. As shown in Fig. 4B, removal of C-terminal sequences either abutting or encompassing the leucine zipper domain of IKK␥ (mutants D1 and D2) had no detectable effect on the formation of IKK␤⅐Tax and IKK␤⅐IKK␥ complexes (lanes 4 and 5, top and middle panels). However, deletion of the N-terminal region of IKK␥ (amino acids 1-100, mutant D3) completely disrupted both of these interactions (lane 6, top and middle panels). We consider these results to be significant, because the three IKK␥ deletion mutants were comparably coexpressed with Tax in the cytoplasmic compartment (Fig. 4B,  lower panels). Furthermore, parallel experiments conducted with Tax-M22 confirmed the specificity of these higher order interactions (Fig. 4B, lanes 7-12). Tax (2 g) as indicated. Ectopic IKK␤ and Tax were isolated from cytoplasmic extracts by immunoprecipitation (IP) with anti-FLAG M2 (top and middle panels) and anti-Tax (lower panel) antibodies, respectively. Resultant immunocomplexes were analyzed for the presence of either Tax or IKK␥ on immunoblots as indicated in the right margin. The position of immunoglobulin heavy chains (H) is indicated. B, S107 cells (1 ϫ 10 7 ) were cotransfected with expression vectors for FLAGtagged IKK␤, IKK␥, and Tax (5 g each) as indicated. Ectopic IKK␤ was immunoprecipitated from cytoplasmic extracts as described above and assayed for IB kinase activity (see Fig. 2A, legend). WT, wild type.
To extend these findings with ectopically expressed IKK␤, we monitored the incorporation of the same IKK␥ mutants into endogenous IKK complexes. For these studies, 293T cells were transfected with IKK␥ and Tax expression vectors, followed by immunoprecipitation of ectopic IKK␥ from the corresponding cytoplasmic extracts. The resultant IKK␥ immunocomplexes were analyzed for the presence of Tax-activated IKKs using an in vitro kinase assay. As shown in Fig. 4C (top panel), overexpression of wild type IKK␥ alone yielded minimal endogenous IKK activity (lane 2). However, coexpression of Tax with IKK␥ led to potent activation (lane 3). Consistent with their ability to target Tax to ectopic IKK␤, IKK␥ mutants D1 and D2 were fully competent to mediate Tax activation of endogenous IKK catalytic activity (lanes 4 and 5). In contrast, the N-terminal deletion mutant of IKK␥ failed to reconstitute a functional Tax⅐IKK signaling axis (lane 6). We conclude that the N terminus of IKK␥ is required for stable integration of this subunit into endogenous IKK, which in turn renders the holoenzyme susceptible to persistent activation by Tax.
In summary, we have found that the IKK␥ subunit of TNFresponsive IKKs is an essential core component of Tax-associated IKKs in HTLV-1-infected T cells. Higher order complexes containing Tax, IKK␥, and the IKK␣⅐IKK␤ catalytic subunits are highly resistant to dissociation in vitro, underscoring the specificity of this pathologic viral/host interaction. In vivo reconstitution experiments demonstrate that IKK␥ directs the assembly of Tax⅐IKK␤ complexes, resulting in the persistent expression of IB kinase activity. Thus, IKK␥ functions in Tax-mediated IKK activation at the level of Tax⅐IKK docking. By analogy, this targeting mechanism may reflect an important role for IKK␥ in coupling TNF-responsive IKKs to upstream physiologic activators, such as NIK and MEKK1 (31).