Role of Akt in the growth of AML-M5 cells and in the de-repression of N-CoR target gene.

<p><i>A,</i> Akt is linked to the growth of AML-M5 cells. Growth of various AML-M5 (THP-1, Nomo-1, MM-1, MV-4–11 and SigM5) and non-AML-M5 (U937, K562 and HL-60) cells treated with Akti-X, a selective inhibitor of Akt, was determined. Asterisk indicates p<0.05. <i>B</i>, Akt inhibition restores N-CoR mediated <i>Flt3</i> gene repression. Level of <i>Flt3</i> transcripts in THP-1 cells treated with AEBSF, genistein or Akt siRNA was determined by RT-PCR. Transcript and protein levels of N-CoR, Akt and pAkt (Ser473) were also determined. <i>C,</i> Loss of N-CoR mediated repression of <i>Flt3</i> due to misfolding. The effect of wild type N-CoR (WT) or the spontaneously misfolded N-CoR (phosphomimetic S1450E) on the <i>Flt3</i> promoter was determined by luciferase assay (top panel). The amount of N-CoR transfected was determined by western blotting assay with flag antibody (bottom panel). <i>D,</i> Schematic representation of transcriptional control imparted by native and misfolded N-CoR. During cellular differentiation, the N-CoR containing co-repressor complex occupies the promoter of growth promoting N-CoR target genes such as <i>Flt3,</i> effectively blocking their expression (left panel). In AML-M5 cells, misfolded conformational dependent loss of N-CoR due to Akt mediated phosphorylation results in the dissociation of the co-repressor complex from the promoter of N-CoR target genes, thus allowing the transcriptional activators to access the promoter and activate the gene (right panel).