Serine 1450 phosphorylation is critical for Akt-induced N-CoR misfolding.

<p><i>A</i>, Serine to alanine substitution at 1450 abrogated Akt-induced N-CoR misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450A or T1925A mutants) in 293T cells transfected with the constitutively active myr-Akt was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions were separated by high-speed centrifugation and N-CoR levels in each fraction were determined with flag antibody (upper panel). The relative solubility/insolubility of β-actin in each fraction was determined as a control (middle panel). The level of total of protein in each fraction was determined by coomassie blue staining (lower panel). <i>B,</i> N-CoR S1450E, the phosphomimetic mutant of N-CoR, displayed signs of misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450E) in 293T cells was determined by protein solubility assay. <i>C,</i> S1450E is localized in cytosol. Subcellular distribution of flag-tagged N-CoR (WT, S1450A and S1450E) was determined by confocal microscopy after staining of the cells transfected with each plasmid with flag antibody. <i>D,</i> S1450E can induce ER stress. Level of GRP78/BiP (green) in 293T cells transfected with S1450E (red) was determined by confocal microscopy after staining the cells with flag and GRP78/BiP antibodies.