Tax oncoprotein induces both poly- and monoubiquitination of FoxO4. Flag-FoxO4 was coexpressed with His-Ubi and various amounts of Tax in HEK293T cells. At 48 h after transfection, the cells were treated with 10 μM MG132 for 3 h. (A) Subsequently, whole-cell extracts (WCE) were prepared from a 10% fraction of the cells and analyzed by Western blotting (WB) using anti-FoxO4, anti-Tax, and or anti-α-tubulin antibodies. (B) The remaining 90% of the cells were lysed under denaturing conditions, and ubiquitinated proteins were precipitated using Ni-NTA agarose. Purified ubiquitinated proteins were analyzed by Western blotting for the presence of ubiquitinated forms of Flag-FoxO4 (Ubi-FoxO4) using anti-FoxO4 antibodies. Whole-cell extracts from Flag-FoxO4-expressing HEK-293T cells (WCE-Flag-FoxO4) were loaded on the gel for size comparison. (C) HA-FoxO4 was coexpressed with His-Ubi and various amounts of Flag-Tax in HEK293T cells. Ni-NTA pulldown assays were performed as described for panel B. HA-FoxO4 and Flag-Tax expression was verified by Western blotting of whole-cell extracts using anti-HA, anti-Flag, or anti-α-tubulin antibodies, and ubiquitinated proteins were analyzed by Western blotting for the presence of ubiquitinated forms of HA-FoxO4 (Uib-FoxO4) using anti-FoxO4 antibodies. (D) HA-FoxO4 was coexpressed in HEK293T cells with Flag-Tax and a mutant of ubiquitin in which all seven lysine residues have been mutated to arginines (His-Ubi K7R). Ni-NTA pulldown assays were performed as described for panel B. HA-FoxO4 and Flag-Tax expression was verified by Western blotting of whole-cell extracts using anti-HA, anti-Flag, or anti-α-tubulin antibodies, and ubiquitinated proteins were analyzed by Western blotting for the presence of ubiquitinated forms of HA-FoxO4 (Ubi-FoxO4) using anti-FoxO4 antibodies.

Tax enhances FoxO4/MDM2 interaction. Flag-FoxO4 was coexpressed with Myc-MDM2 and various amounts of Tax in HEK293T cells. At 48 h after transfection, the cells were treated with 10 μM MG132 for 3 h. (A) Subsequently, whole-cell extracts (WCE) were prepared from a 10% fraction of the cells and analyzed by Western blotting using anti-Flag, anti-Myc, anti-Tax, or anti-α-tubulin antibodies. (B) The remaining 90% of the cells were subjected to immunoprecipitation (IP) with anti-Flag antibodies. Immunoprecipitated proteins were then fractionated by SDS-PAGE and revealed by Western blotting for the presence of Myc-MDM2 and Flag-FoxO4 using anti-Myc and anti-Flag antibodies, respectively.

Tax oncoprotein induces degradation and phosphorylation of endogenous FoxO4 in Jurkat leukemia cells. Jurkat leukemia cells were cotransfected with 1 μg of HTLV-1 LTR-Luc vector and various amounts (μg) of the Tax expression vector pMT2-Tax. (A) Subsequently, whole-cell extracts were prepared and analyzed by Western blotting (WB) using anti-FoxO4 detecting total FoxO4 and anti-α-tubulin antibodies. Luciferase activities directed by the HTLV-1 LTR promoter were used to monitor Tax expression. (B) Jurkat leukemia cells were transfected with various amounts (μg) of the Tax expression vector pMT2-Tax. Protein extracts were prepared and analyzed by Western blotting using anti-phospho-FoxO4 and anti-α-tubulin antibodies. (C) Whole-cell extracts were prepared from Jurkat cells, HTLV-1-transformed MT4 and C8166 cells, Jurkat cells transfected with 20 μg Flag-Tax, and HEK293T cells transfected with 100 ng of Flag-Tax plus 200 ng of Flag-FoxO4 expression vector. Proteins were then analyzed by Western blotting using anti-Tax, anti-FoxO4, and anti-α-tubulin antibodies.

Tax oncoprotein induces FoxO4 degradation through the proteasome pathway. (A) HEK293T cells were transfected with 100 ng of a vector expressing the transcription factor FoxO4 (Flag-FoxO4) and various amounts of a vector expressing the viral transactivator Tax (Flag-Tax). For each experimental condition, the relative amounts of Tax-transfected versus FoxO4-transfected cells (Tax/FoxO4 ratios) are indicated. At 48 h after transfection, the cells were not treated (−MG132) or treated (+MG132) with 10 μM MG132 for 3 h. Subsequently, whole protein extracts were prepared and analyzed by Western blotting (WB) using anti-FoxO4 (top), anti-Tax (middle), or anti-α-tubulin (bottom) antibodies. FoxO4 cleavage products (FoxO4-CP) are indicated. (B) HEK293T cells were transfected with Flag-Tax and various amounts of Flag-FoxO4. For easier comparison with the experiment presented in panel A, Tax/FoxO4 ratios are indicated. At 48 h after transfection, whole protein extracts were prepared and analyzed as described for panel A. (C) HEK293T cells were transfected with a vector expressing Flag-FoxO4-TM and various amounts of a vector expressing the viral transactivator Tax (Flag-Tax). At 48 h after transfection, whole protein extracts were prepared and analyzed as described for panel A.

Comparison of Tax-mediated ubiquitination of FoxO4 and FoxO4-TM. Flag-FoxO4 and Flag-FoxO4-TM were coexpressed with His-Ubi and various amounts of Tax in HEK293T cells. At 48 h after transfection, the cells were treated with 10 μM MG132 for 3 h. (A) Subsequently, whole-cell extracts (WCE) were prepared from a 10% fraction of the cells and analyzed by Western blotting (WB) using anti-FoxO4, anti-Tax, or anti-α-tubulin antibodies. (B) The remaining 90% of the cells were lysed under denaturing conditions, and ubiquitinated proteins were precipitated using Ni-NTA agarose. Purified ubiquitinated proteins were analyzed by Western blotting for the presence of ubiquitinated forms of Flag-FoxO4 and Flag-FoxO4-TM (Ub-FoxO4) using anti-FoxO4 antibodies. (C) As a control for ubiquitination reactions, the presence of ubiquitinated forms of Tax was analyzed using anti-Tax antibodies.

MDM2-targeting short hairpin RNA antagonizes Tax-mediated FoxO4 ubiquitination. (A) HEK293T cells were cotransfected with a vector expressing His-MDM2 and a vector expressing either a nontargeting control shRNA (Scr) or a vector expressing MDM2-targeting shRNA (sh1, sh2, sh3, sh4, or sh5). Subsequently, whole-cell extracts (WCE) were prepared and analyzed by Western blotting (WB) using anti-MDM2 and anti-α-tubulin antibodies. (B) Flag-FoxO4 was coexpressed with His-Ubi and various amounts of a vector expressing Tax in the presence of a vector expressing either a nontargeting control shRNA (shRNA-Scr) or a mix of MDM2-targeting sh1 and sh5 shRNAs (shRNA-MDM2) in HEK293T cells. At 48 h after transfection, the cells were treated with 10 μM MG132 for 3 h. Subsequently, whole-cell extracts were prepared from a 10% fraction of the cells and analyzed by Western blotting using anti-MDM2, anti-FoxO4, anti-Tax, or anti-α-tubulin antibodies. (C) The remaining 90% of the cells were lysed under denaturing conditions, and ubiquitinated proteins were precipitated using Ni-NTA agarose. Purified ubiquitinated proteins were analyzed by Western blotting for the presence of ubiquitinated forms of Flag-FoxO4 (Ub-FoxO4) using anti-FoxO4 antibodies.

Tax antagonizes FoxO4 transcriptional activity. (A) HEK293T cells were cotransfected with the pGADD45-Luc reporter plasmid alone or with HA-FoxO4 expression vector and various amounts of pMT2-Tax expression vector. At 48 h posttransfection, luciferase activities were quantified. Results from two independent experiments performed in duplicate are presented. (B) Protein extracts were analyzed by Western blotting (WB) using anti-FoxO4 and anti-α-tubulin antibodies.