1. Volume 93, Issue 5, Pages 875-884 (May 1998)
HTLV-I Tax Protein Binds to MEKK1 to Stimulate IκB Kinase Activity and NF-κB Activation 
Min-Jean Yin, Lori B. Christerson, Yumi Yamamoto, Youn-Tae Kwak, Shuichan Xu, Frank Mercurio, Miguel Barbosa, Melanie H. Cobb, Richard B. Gaynor 
Cell 
Volume 93, Issue 5, Pages (May 1998)
DOI: /S (00)

2. Figure 1 Tax Activation of NF-κB Is Mediated by IKKβ
(A) Jurkat cells were transfected with an HIV-1 LTR CAT plasmid (0.25 μg) in the presence or absence of Tax (3 μg) and different quantities of either IKKα (K→M) and (SS→AA) or IKKβ (K→M) and (SS→AA) dominant negative cDNA constructs as labeled. The fold of Tax transactivation is the increase of HIV-1 LTR CAT activity in the presence of Tax as compared to its absence. Western blot analysis with either the M2 Flag or 12CA5 monoclonal antibody using extracts prepared from each transfections is shown.
(B) Jurkat cells were transfected with the HIV-1 LTR CAT plasmid with different quantities of IKKα or IKKβ dominant negative cDNAs in either the presence or absence of TNFα (20 ng/ml). CAT activity was determined from either untreated or TNFα-treated cells and was used to calculate the fold of TNFα activation.
(C) Jurkat cells were transfected with an HTLV-I LTR CAT plasmid in the presence of Tax and different amounts of dominant negative IKKα or IKKβ cDNAs. CAT activity and the fold transactivation was calculated as in (A).
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3. Figure 2 Tax Regulates IKKβ Phosphorylation of IκBα
(A) COS cells were transfected with IKKβ (0.1 μg) alone (lane 1); with TNFα (20 ng/ml) for 10 min prior to harvest (lane 2); or in the presence of wild-type tax (lane 3), the M22 (lane 4), or the M47 tax (lane 5) cDNAs as indicated and harvested 24 hr later. Kinase assays were performed using a wild-type (lanes 1–5) or a serine 32/36 mutant (lanes 6–10) of GST-IκBα (amino acids 1–54) as a substrate. Western blot analysis from each transfection was performed with the M2 Flag monoclonal antibody.
(B) Extract was prepared from Jurkat cells, the HTLV-I-transformed T cell line SLB, the Jurkat cell line JPX-9 in either the absence (lane 3) or presence of 20 μM cadmium (lane 4), Jurkat cells in the presence of cadmium (lane 5), or COS cells transfected with IKKβ (lane 6). Immunoprecipitation with a polyclonal antibody that recognizes IKKα/IKKβ was performed followed by kinase assays using GST-IκBα (amino acids 1–54) as a substrate. In the bottom panels, Western blot analysis was performed with either the rabbit polyclonal antibody directed against IKK or a monoclonal antibody directed against Tax using the extracts assayed in the kinase assays in lanes 1–4 of the upper panel.
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4. Figure 3 MEKK1 Is Critical for Tax Activation of IKKβ
(A) Jurkat cells were transfected with the HIV-1 LTR CAT plasmid (0.25 μg) in either the presence or absence of tax (3 μg) and 6 μg of the different dominant negative constructs as indicated. CAT assays were performed, and the fold of Tax activation was calculated as described. Western blots using cell lysates from each transfection are shown in the bottom panel.
(B) Jurkat cells were transfected with an HIV-1 LTR CAT plasmid and different quantities of various dominant negative mutant cDNAs as indicated in either the presence or absence of TNFα. CAT activity was determined, and the fold of activation by TNFα was calculated. Western blot analysis of the cell lysates from each transfection containing 6 μg of the different dominant negative mutants is shown in bottom panel.
(C) Jurkat cells were transfected with a wild-type or NF-κB mutated HIV-1 LTR CAT construct alone or in the presence of MEKK1, tax, or both constructs. The fold of Tax and MEKK1 activation was calculated.
(D) Jurkat cells were transfected with an HIV-1 LTR CAT construct, MEKK1, and different amounts of dominant negative IKKα and IKKβ mutants as indicated. CAT assays were performed and the fold of MEKK1 activation was calculated.
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5. Figure 4
Dominant Negative MEKK1 Prevents Tax Stimulation of IκBβ Kinase Activity and the Nuclear Translocation of NF-κB
(A) An IKKβ cDNA (0.1 μg) was transfected into COS cells either alone (lane 1), in the presence of TNFα (lane 2), or with the various constructs as indicated. IKKβ was immunoprecipitated with M2 Flag monoclonal antibody followed by kinase assays as described. Western blot analysis of each lysate was performed with the M2 Flag monoclonal antibody.
(B) An IKKα cDNA (2 μg) was transfected into COS cells alone (lane 1), in the presence of TNFα (lane 2), or with the various constructs as labeled. IKKα was immunoprecipitated with the 12CA5 monoclonal antibody and kinase assays were performed as described. Western blot analysis of the lysates was performed using the 12CA5 monoclonal antibody (lanes 1–5).
(C) COS cells were transfected with the different cDNA constructs as indicated. Nuclear extract (20 μg) was used in gel retardation assays with an NF-κB (upper panel) or an Sp1 (lower panel) oligonucleotide as probe. Probe alone is shown in lane 1, the extracts from the different transfections are shown in lanes 2–5, and Jurkat cell nuclear extract (lane 6) alone or prepared following TPA-treatment (lane 7) is shown as a control for NF-κB nuclear translocation.
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6. Figure 5 Tax and MEKK1 Regulate IKKβ Kinase Activity
(A) An influenza hemagglutinin-tagged MEKK1 cDNA (0.1 μg) was transfected into COS cells with a pCMV5 vector alone (lane 1) or vectors containing wild-type tax (lane 2), the M22 tax mutant (lane 3), or the M47 tax mutant (lane 4). MEKK1 was immunoprecipitated with 12CA5 monoclonal antibody followed by kinase assays (lanes 1–4). Recombinant MEKK1 is shown in lane 5. Western blot analysis of each of the cell lysates with 12CA5 antibody is shown in the bottom panel (lanes 1–4).
(B) COS cells that were transfected with vector alone (lane 1) or transfected with MEKK1 in either the absence (lane 2) or presence of Tax (lane 3) were immunoprecipitated with the 12CA5 monoclonal antibody and assayed for kinase activity in the presence of the GST-MEK4 substrate. Recombinant MEKK1 and GST-MEK4 (lane 4) or GST-MEK4 alone (lane 5) were also assayed.
(C) COS cells were transfected with wild-type (lanes 1, 2, 5, and 6) or mutant (lanes 3, 4, 7, and 8) IKKα and IKKβ constructs as above, and these proteins were immunoprecipitated from the cell lysates. The immunoprecipitates were then preincubated in either the presence (lanes 1, 3, 5, 7, and 9) or the absence (lanes 2, 4, 6, 8, and 10) of the recombinant catalytic subunit of MEKK1 and cold ATP. Kinase assays in the presence of [γ-32P]ATP were then performed with either GST-IκBα (lanes 1–8) or GST-MEK4 (lanes 9–10) followed by SDS-PAGE and autoradiography.
(D) The baculovirus-produced and -purified kinase-deficient mutant IKKβ/K44→M was used as a substrate in an in vitro kinase assay either alone (lane 1) or in the presence of either a MEKK1 catalytic domain (lane 2) or a full-length MEKK1 protein (lane 3). In addition, the MEKK1 catalytic domain (lane 4), the MEKK1 full-length protein (lane 5), or constitutively active IKKβ/SS→EE mutant (lane 6) were each assayed alone.
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7. Figure 6 Tax and MEKK1 Directly Interact In Vivo
(A) HeLa cells were transfected with a pTM1 expression vector alone or a vector containing various cDNAs as indicated followed by infection with a recombinant vaccinia virus producing T7 polymerase. Cells were harvested at 36–48 hr, and the lysates were first immunoprecipitated with Tax antibody and then analyzed by Western blot analysis (top panel) using either NIK (lanes 1–4) or MEKK1 (lanes 5–13) polyclonal antibody. Western blot analysis of each cell lysate was performed with monoclonal antibody against Tax (middle panel) or polyclonal antibody against either NIK or MEKK1 (bottom panel).
(B) Extract was prepared from Jurkat cells (lanes 1, 6, and 11), SLB cells (lanes 2, 7, and 12), HeLa cells transfected with vector alone (lanes 3, 8, and 13), or HeLa cells transfected with a pTM1 vector expressing either tax (lanes 4, 9, and 14) or MEKK1 (lanes 5 and 10) followed by infection with a recombinant vaccinia virus expressing T7 polymerase. Immunoprecipitation was performed by first immunoprecipitating with antibody directed against either Tax (lanes 1–4 and 6–9) or Myc (lanes 11–14) followed by the addition of [γ-32P]ATP, boiling the immunoprecipitates, and performing a second immunoprecipitation with either β-galactosidase antibody (lanes 1–4) or MEKK1 antibody (lanes 6–9 and 11–14). In lanes 5 and 10, lysates containing transfected MEKK1 were labeled with [γ-32P]ATP and then immunoprecipitated directly with either β-galactosidase (lane 5) or MEKK1 (lane 10) antibodies.
(C) HeLa cells were transfected with pTM1 vectors containing wild-type tax (lane 1), the M22 tax mutant (lane 2), the M47 tax mutant (lane 3), or MEKK1 alone (lane 4). Immunoprecipitation was performed with the Tax monoclonal antibody (lanes 1–3) followed by the addition of [γ-32P]ATP, boiling of the immunoprecipitates, and a second immunoprecipitation with MEKK1 antibody. The MEKK1-transfected cell lysate was labeled with [γ-32P]ATP and directly immunoprecipitated with MEKK1 antibody (lane 4). In the bottom panel, the lysates used in the immunoprecipitation were analyzed by Western blot analysis with Tax antibody.
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8. Figure 7 In Vitro Interactions of Tax and MEKK1
(A) Wild-type Tax and the Tax M22 and M47 mutant proteins containing a carboxy-terminal protein kinase A substrate site were 32P-labeled by the addition of the PKA catalytic subunit (Yin et al. 1995). The 32P-labeled wild-type Tax protein (20% of input shown in lane 1) was analyzed by incubation with GST alone (lane 2) or GST fusions of different portions of MEKK1 as labeled.
(B) Either 32P-labeled wild-type Tax (lanes 1–3), the Tax M47 mutant (lanes 4–6), or the Tax M22 mutant (lanes 7–9) was bound to the indicated GST fusion proteins followed by SDS-PAGE and autoradiography. Twenty percent of the input protein is shown in lanes 1, 4, and 7; binding to GST beads is shown in lanes 2, 5, and 8; and binding to GST-MEKK1 (30–220) is shown in lanes 3, 6, and 9.
(C) A schematic of the GST-MEKK1 fusion proteins used to analyze Tax binding.
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