Deacetylation by SIRT1 promotes the tumor-suppressive activity of HINT1 by enhancing its binding capacity for β-catenin or MITF in colon cancer and melanoma cells

摘要

Flag-HINT1 was coexpressed with all a or each b of the indicated HATs, p300, CBP, Tip60, pCAF, and Gcn5, in HEK293T cells, and then cells were treated with nicotinamide (NAM, 10 mM for 12 h) and trichostatin A (TSA, 1 µM for 12 h). Acetylation levels of Flag-bead-immunoprecipitated (IPed) HINT1 were determined by immunoblot analysis using pan-acetyl lysine (Ack) antibody. For the reciprocal interaction between HINT1 and CBP, Flag-HINT1 was coexpressed with HA-CBP in HEK293T cells. HINT1 or CBP was immunoprecipitated with HA c or Flag d antibody, respectively, following western blot analysis using antibodies as indicated. e Endogenous HINT1 or CBP proteins in DLD1 or SW480 cells were immunoprecipitated and immunoblotted with antibodies as indicated. f CBP was coexpressed with each of the Flag-HINT1 wild-type versions or mutants (K21R, K30R, K21/30R; lysine to arginine) in HEK293T cells. Acetylation levels of Flag-bead-IPed HINT1 were examined by immunoblot analysis. g Sequence alignment of the putative acetylation sites K21 and K30 in HINT1 from different species. h Schematic representation of the positions of putative acetylation sites K21 and K30 within HINT1.