Influence on tumor growth by acetylation state of HSP27

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Using silencing of HDAC6 or NACC1 on MB-231, hyperacetylation of HSP27 was seen by immunoprecipitation of the endogenous proteins with anti-HSP27 antibody, but not with anti-acetyl-lysine antibody. None of serial mutations from lysine to arginine in HSP27 also led to evident change of the acetylation state by treatment with HDAC6 siRNA or TSA (HDAC inhibitor). Additionally, LC tandem mass spectorometric analysis did not detect acetylation sites of HSP27 on treatment with HDAC6 siRNA or TSA. Therefore association between HSP27 and β-catenin was examined. Immunohistochemical analysis indicated colocalization of HSP27 and β-catenin in the cytoplasm of MB-231. The interaction between HSP27 and β-catenin was confirmed by immunoprecipitation using cell lysate of MB-231and pull down assay using Halo- or Flag-tagged proteins of HSP27 and β-catenin by wheat germ protein expression system . Silencing of HSP27 induced the ubiquitination ofβ-catenin and the degradation by proteasome system.