Akt-mediated regulation of Notch-dependent transcription

Abstract

We have investigated the effect of Akt/PKB on Notch intracellular domain (NICD)-mediated transcription. The transient transfection studies using both 293T and Cos7 cells revealed that myristylated constitutive active Akt could down-regulate NICD-dependent transcription in both cell lines. Studies using kinase-dead dominant negative Akt (dnAkt - Akt1 K179M, Akt2 K180A, and Akt3 K177M) showed that Akt kinase activity is responsible for down-regulation of NICD-dependent transcription in all three different reporter constructs (4XCSL, HES-Luc, 6XNRE). Studies using histone deacetylase, HDAC1, dominant negative HDAC1(H141A) and TSA revealed that regulation of Notch-dependent transcription is not mediated by HDAC1 dependent mechanism. Co-immunoprecipitation studies with Flag-CBF1 and SMRT showed that Akt could not affect the association between CBF1 and SMRT, suggesting that recruitment of SMRT is not the mechanism responsible for Akt-mediated downregulation of Notch-dependent transcription. When the intracellular localization of myc-tagged NICD was investigated, all three Akt isoforms effectively inhibited proper nuclear localization of NICD. Futhermore, dnAkt isoforms could not efficiently inhibit nuclear localization of NICD, suggesting that kinase activity of Akt is responsible for mislocalization of NICD in the perinuclear cytoplasm. Studies using Akt substrate antibody which recognizes peptides and proteins containing RXRXXS/T motif phosphorylated by Akt, showed that there is an increase in the phosphorylation of NICD by Akt coexpression. Taken together, these results may suggest possible regulation of NICD transcription activity by Akt-mediated phosphorylation and subsequent inhibition of proper nuclear localization of NICD.