Tax and TRAF6 promote MCL-1 K63-linked polyubiquitination on C-terminal lysines.

<p>(A) Schematic of MCL-1 lysine mutants. The substituted arginine (R) residues are highlighted in blue. (B) Requirement of the four C-terminal lysine residues in Tax and TRAF6-induced MCL-1 stabilization. Immunoblotting was performed with whole cell lysates of 293 cells transfected with the indicated Flag-MCL-1 plasmids together with Tax or Flag-TRAF6. (C) Identification of C-terminal lysines required for MCL-1 stabilization. Immunoblotting was performed with whole cell lysates of 293 cells either transfected with the indicated Flag-MCL-1 plasmids together with Tax (top two panels) or treated with etoposide for 24 h after transfection (bottom two panels). (D) Ub-independent proteasomal degradation of MCL-1. Immunoblotting was performed with whole cell lysates of 293 cells transfected with Flag-MCL-1 WT or All-KR and then treated with etoposide and/or MG-132 (10 µM) for 24 h. (E) Tax and TRAF6 block the interaction between MCL-1 and the 20S proteasome. Immunoblotting was performed with Flag immunoprecipitates and whole cell lysates derived from 293 cells transfected with Flag-MCL-1 or MCL-1-All-KR together with Tax or His-TRAF6, and treated with the DSP cross-linker (2 µM) for 30 min prior to cell lysis. Asterisk (*) indicates immunoglobulin light chain. (F) Tax promotes the K63-linked polyubiquitination of the four C-terminal MCL-1 lysines. K63-Ub assay was performed with lysates from 293 cells transfected with WT 6×His-MCL-1 and the indicated MCL-1 mutants together with Tax. (G) Proposed molecular mechanism of Tax and TRAF6-induced stabilization of MCL-1.