N-CoR is phosphorylated by Akt.

<p><i>A</i>, Two potential Akt consensus motifs were identified in N-CoR. The screen capture of the results of the N-CoR putative kinase recognition motif search in the human protein reference database program is shown. Only the sequences that matched to the Akt substrate motifs in the search are presented (upper panel). The Akt substrate motif and it surrounding sequences are highly conserved in human and mouse N-CoR sequences (lower panel). <i>B,</i> Genetic ablation of Akt abrogated N-CoR phosphorylation. Level of N-CoR protein phosphorylated (upper panel, left) at the Akt consensus motif in THP-1 cells treated with AEBSF or Akt siRNA was determined by staining the immunoprecipitated (IP) full length N-CoR protein (lower panel, left) with phospho-Akt substrate (RXRXX pS/pT) antibody. The levels of N-CoR, pAkt (Ser 473) and Akt in crude cellular extracts used in IP assay were determined (right panel). <i>C</i>, Level of N-CoR protein phosphorylated (upper panel, left) at the Akt consensus site in flag-tagged N-CoR protein immunoprecipitated (lower panel, left) from 293T cells transfected with myr-Akt or control plasmid was determined by western blotting with a phospho-Akt substrate (RXRXX pS/pT) specific antibody. An aliquot of crude cell extract was probed with the respective antibodies to determine the levels of flag-N-CoR, pAkt (Ser 473) and Akt (right panel). <i>D,</i> Constitutively active Akt phosphorylates N-CoR <i>in vitro</i>. Affinity purified flag-tagged N-CoR was incubated with purified myr-Akt and the level of phosphorylated N-CoR protein after the incubation was determined by western blotting assay with the phospho-Akt substrate (RXRXX pS/pT) antibody.