PMC

(A) Mass spectrometry was completed on four BRMS1 mutants with full-length BRMS1 (BRMS1^WT), BRMS1 aa 1–229 (BRMS1^1-229), BRMS1 (S237A), BRMS1 (S237D), and BRMS1 aa 230–246 (BRMS1^230-246). (B). Secondary structure prediction of the BRMS1 proteins by I-TASSER, followed by the BRMS1 predicted 3D model (C). The structure generated demonstrates that the C-terminal region consists of helical, coiled, and strand regions. (D) t-SNE analysis was implemented for the analysis of the mutant data. Four clusters are generated by t-SNE. (E) Components of the Sin3/HDAC (in respective cluster colors from Fig 1D) and BRMS1 (in blue) proteins are highlighted.

(A) BRMS1 protein has a shorter half-life in NSCLC than in NHBE cells. NHBE and A549 and H1299 cells were treated with CHX (100 µM) for the indicated times. BRMS1 expression was probed by immunoblot (Upper). The half-life (t_1/2) of BRMS1 protein was calculated (Lower). (B) TNF promotes BRMS1 degradation. NHBE and H1299 cells were transfected with HA-tagged BRMS1. Thirty-six hours posttransfection, pulse-chase analysis was performed with or without TNF (20 ng/mL) at the indicated times. BRMS1 was visualized by autoradiography, and t_1/2 was calculated. (C) Schematic representation of BRMS1 protein. Orange rectangles: Coiled-coil domains; red characters: putative CK2-phosphorylated sites. (D) BRMS1 is phosphorylated by CK2 in vitro. In vitro phosphorylation assays were performed by incubating GST-BRMS1 with recombinant CK2 and subjection to SDS-PAGE gel. Phospho-GST-BRMS1 was visualized by autoradiography. (E) TNF induces BRMS1 phosphorylation at S30. H157 and H1299 cells were treated with TNF (20 ng/mL) for the indicated times. BRMS1 (pS30) was detected by immunoblot. (F) S30A mutant abrogates TNF-induced BRMS1 phosphorylation. H157 cells were transfected with HA-BRMS1 wild-type or S30A mutant and stimulated with or without TNF (20 ng/mL) for an additional 2 h. Immunoprecipitation (IP) assays were performed using anti-HA antibody, and BRMS1 (pS30) was detected.

(A) CK2 mediates TNF-induced BRMS1 nuclear exportation. H1299 cells were pretreated with MG132 (5 µM) alone or with CX4945 (30 µM) for 2 h, followed by TNF (20 ng/mL). Nuclear/cytosolic BRMS1 expression was probed. (B) S30A mutant abrogates TNF-induced nuclear exportation of BRMS1. H157 cells were transfected with HA-tagged BRMS1 wild-type or S30A mutant. Transfected cells were treated with or without TNF (20 ng/mL) for 2 h; HA-BRMS1 in nuclear extract (NE) or cytosolic extract (CE) was probed separately. (C) TNF enhances the interaction of BRMS1 and 14-3-3ε. H157 cells were pretreated with MG132 (5 µM) for 2 h and then treated with or without TNF (20 ng/mL) for an additional 2 h. Coimmunoprecipitation (IP) assays were performed using the indicated antibodies. The presence of 14-3-3ε was detected. (D) S30A mutant abrogates BRMS1 binding to 14-3-3ε. H1299 cells were transfected with HA-tagged BRMS1 wild-type or S30A. Co-IP assays using NE were performed, and the presence of 14-3-3ε was probed. (E) BRMS1 is primarily located in the nucleus following loss of 14-3-3ε. Immunofluorescent merged micrographs show subcellular localization of BRMS1 (red)/DAPI (blue) in H1993 cells infected with shRNA 14-3-3ε or scramble. Western blots indicate that 14-3-3ε was specifically knocked down. (F) Knockdown 14-3-3ε inhibits TNF-induced nuclear exportation of BRMS1. H157 cells were infected with shRNA 14-3-3ε or scramble, pretreated with MG132 (5 µM) for 2 h, and then treated with or without TNF (20 ng/mL) for an additional 2 h. HA-BRMS1 in NE or CE was detected.

(A) CX4945 abrogates TNF-induced BRMS1 degradation. H1299 cells were pretreated with or without CX4945 (30 µM) for 2 h, followed by TNF (20 ng/mL) for 16 h. The indicated proteins were evaluated. (B) CK2α' induces BRMS1 degradation. H157 cells were transfected with the indicated CK2 subunits. Protein levels of BRMS1 and CK2s were evaluated. BRMS1 expression was quantified by densitometry (normalized with tubulin) and labeled under each corresponding band. (C) CK2α', but not α, interacts with BRMS1. H1299 cells were transfected with HA-tagged CK2α, α', or empty vector. Coimmunoprecipitation (IP) assays were performed following treatment with MG132 (5 µM) for 16 h, and BRMS1 was detected. (D) Knockdown CK2α' inhibits TNF-induced BRMS1 degradation. H1299 cells were transfected with siRNA CK2α' or scramble and treated with TNF (20 ng/mL). The indicated proteins were evaluated. (E) CK2α' enhances BRMS1 polyubiquitination. NSCLC cells were transfected with HA-CK2α' and treated with MG132 (5 µM) for 16 h. Ubiquitination assays were conducted. (F) S30A mutation abrogates CK2α'-induced BRMS1 degradation. H1299 cells were cotransfected with HA-BRMS1 wild-type or S30A, and HA-CK2α'. The indicated proteins were assessed.