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Akt-induced phosphorylation of N-CoR at serine 1450 contributes to its misfolded conformational dependent loss (MCDL) in acute myeloid leukemia of the M5 subtype.

Nin DS, Ali AB, Okumura K, Asou N, Chen CS, Chng WJ, Khan M - PLoS ONE (2013)

Bottom Line: N-CoR displayed significantly higher level of serine specific phosphorylation in almost all AML-M5 derived cells and was subjected to processing by AML-M5 specific aberrant protease activity.Consistent with this finding, a constitutively active Akt consistently phosphorylated N-CoR leading to its misfolding; while the therapeutic and genetic ablation of Akt largely abrogated the MCDL of N-CoR in AML-M5 cells.The N-CoR misfolding pathway could be the common downstream thread of pleiotropic Akt signaling activated by various oncogenic insults in some subtypes of leukemia and solid tumors.

View Article: PubMed Central - PubMed

Affiliation: Cancer Science Institute of Singapore, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

ABSTRACT
The nuclear receptor co-repressor (N-CoR) is a key component of the generic co-repressor complex that plays an important role in the control of cellular growth and differentiation. As shown by us recently, the growth suppressive function of N-CoR largely relies on its capacity to repress Flt3, a key regulator of cellular gorwth during normal and malignant hematopoesis. We further demonstrated how de-repression of Flt3 due to the misfolded conformation dependent loss (MCDL) of N-CoR contributed to malignant growth in acute myeloid leukemia (AML). However, the molecular mechanism underlying the MCDL of N-CoR and its implication in AML pathogenesis is not fully understood. Here, we report that Akt-induced phosphorylation of N-CoR at the consensus Akt motif is crucial for its misfolding and subsequent loss in AML (AML-M5). N-CoR displayed significantly higher level of serine specific phosphorylation in almost all AML-M5 derived cells and was subjected to processing by AML-M5 specific aberrant protease activity. To identify the kinase linked to N-CoR phosphorylation, a library of activated kinases was screened with the extracts of AML cells; leading to the identification of Akt as the putative kinase linked to N-CoR phosphorylation. Consistent with this finding, a constitutively active Akt consistently phosphorylated N-CoR leading to its misfolding; while the therapeutic and genetic ablation of Akt largely abrogated the MCDL of N-CoR in AML-M5 cells. Site directed mutagenic analysis of N-CoR identified serine 1450 as the crucial residue whose phosphorylation by Akt was essential for the misfolding and loss of N-CoR protein. Moreover, Akt-induced phosphorylation of N-CoR contributed to the de-repression of Flt3, suggesting a cross talk between Akt signaling and N-CoR misfolding pathway in the pathogenesis of AML-M5. The N-CoR misfolding pathway could be the common downstream thread of pleiotropic Akt signaling activated by various oncogenic insults in some subtypes of leukemia and solid tumors.

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Role of Akt in the growth of AML-M5 cells and in the de-repression of N-CoR target gene.A, 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. B, Akt inhibition restores N-CoR mediated Flt3 gene repression. Level of Flt3 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. C, Loss of N-CoR mediated repression of Flt3 due to misfolding. The effect of wild type N-CoR (WT) or the spontaneously misfolded N-CoR (phosphomimetic S1450E) on the Flt3 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). D, 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 Flt3, 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).
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pone-0070891-g007: Role of Akt in the growth of AML-M5 cells and in the de-repression of N-CoR target gene.A, 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. B, Akt inhibition restores N-CoR mediated Flt3 gene repression. Level of Flt3 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. C, Loss of N-CoR mediated repression of Flt3 due to misfolding. The effect of wild type N-CoR (WT) or the spontaneously misfolded N-CoR (phosphomimetic S1450E) on the Flt3 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). D, 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 Flt3, 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).

Mentions: To define the role of Akt in the growth of AML-M5 cells, we next investigated the effect of Akti-X on the growth of AML-M5 cells. Akti-X inhibited the growth of multiple AML-M5 derived cells in a dose dependent manner while its effect on non-AML-M5 cells was minimal (Fig. 7A). As shown by us recently, the growth promoting effect of misfolded N-CoR is partly mediated by the de-repression of Flt3, a transcriptional target of N-CoR [11]. Therefore, to test whether Akt promoted the growth of AML-M5 cells through Flt3 de-repression, level of Flt3 transcript in THP-1 cells was measured after siRNA-mediated Akt ablation. As shown in Figure 7B, Akt ablation led to a significant down regulation of Flt3 level in THP-1 cells, possibly through the restoration of native N-CoR conformation and function as observed with genistein (Fig. 7B, left panel). Moreover, the constitutively misfolded phosphomimetic mutant of N-CoR, N-CoR S1450E, could not repress the luciferase reporter activity driven by Flt3 promoter as efficiently as the wild type N-CoR did (Fig. 7C, left panel), suggesting a link between N-CoR phosphorylation and loss of N-CoR’s repressor function. These results suggest that Akt-induced phosphorylation of N-CoR compromised its function as a transcriptional repressor, leading to the ectopic reactivation of growth promoting genes like Flt3, ultimately contributing to malignant growth and transformation of AML-M5 cells (Fig. 7D).


Akt-induced phosphorylation of N-CoR at serine 1450 contributes to its misfolded conformational dependent loss (MCDL) in acute myeloid leukemia of the M5 subtype.

Nin DS, Ali AB, Okumura K, Asou N, Chen CS, Chng WJ, Khan M - PLoS ONE (2013)

Role of Akt in the growth of AML-M5 cells and in the de-repression of N-CoR target gene.A, 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. B, Akt inhibition restores N-CoR mediated Flt3 gene repression. Level of Flt3 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. C, Loss of N-CoR mediated repression of Flt3 due to misfolding. The effect of wild type N-CoR (WT) or the spontaneously misfolded N-CoR (phosphomimetic S1450E) on the Flt3 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). D, 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 Flt3, 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).
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pone-0070891-g007: Role of Akt in the growth of AML-M5 cells and in the de-repression of N-CoR target gene.A, 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. B, Akt inhibition restores N-CoR mediated Flt3 gene repression. Level of Flt3 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. C, Loss of N-CoR mediated repression of Flt3 due to misfolding. The effect of wild type N-CoR (WT) or the spontaneously misfolded N-CoR (phosphomimetic S1450E) on the Flt3 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). D, 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 Flt3, 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).
Mentions: To define the role of Akt in the growth of AML-M5 cells, we next investigated the effect of Akti-X on the growth of AML-M5 cells. Akti-X inhibited the growth of multiple AML-M5 derived cells in a dose dependent manner while its effect on non-AML-M5 cells was minimal (Fig. 7A). As shown by us recently, the growth promoting effect of misfolded N-CoR is partly mediated by the de-repression of Flt3, a transcriptional target of N-CoR [11]. Therefore, to test whether Akt promoted the growth of AML-M5 cells through Flt3 de-repression, level of Flt3 transcript in THP-1 cells was measured after siRNA-mediated Akt ablation. As shown in Figure 7B, Akt ablation led to a significant down regulation of Flt3 level in THP-1 cells, possibly through the restoration of native N-CoR conformation and function as observed with genistein (Fig. 7B, left panel). Moreover, the constitutively misfolded phosphomimetic mutant of N-CoR, N-CoR S1450E, could not repress the luciferase reporter activity driven by Flt3 promoter as efficiently as the wild type N-CoR did (Fig. 7C, left panel), suggesting a link between N-CoR phosphorylation and loss of N-CoR’s repressor function. These results suggest that Akt-induced phosphorylation of N-CoR compromised its function as a transcriptional repressor, leading to the ectopic reactivation of growth promoting genes like Flt3, ultimately contributing to malignant growth and transformation of AML-M5 cells (Fig. 7D).

Bottom Line: N-CoR displayed significantly higher level of serine specific phosphorylation in almost all AML-M5 derived cells and was subjected to processing by AML-M5 specific aberrant protease activity.Consistent with this finding, a constitutively active Akt consistently phosphorylated N-CoR leading to its misfolding; while the therapeutic and genetic ablation of Akt largely abrogated the MCDL of N-CoR in AML-M5 cells.The N-CoR misfolding pathway could be the common downstream thread of pleiotropic Akt signaling activated by various oncogenic insults in some subtypes of leukemia and solid tumors.

View Article: PubMed Central - PubMed

Affiliation: Cancer Science Institute of Singapore, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

ABSTRACT
The nuclear receptor co-repressor (N-CoR) is a key component of the generic co-repressor complex that plays an important role in the control of cellular growth and differentiation. As shown by us recently, the growth suppressive function of N-CoR largely relies on its capacity to repress Flt3, a key regulator of cellular gorwth during normal and malignant hematopoesis. We further demonstrated how de-repression of Flt3 due to the misfolded conformation dependent loss (MCDL) of N-CoR contributed to malignant growth in acute myeloid leukemia (AML). However, the molecular mechanism underlying the MCDL of N-CoR and its implication in AML pathogenesis is not fully understood. Here, we report that Akt-induced phosphorylation of N-CoR at the consensus Akt motif is crucial for its misfolding and subsequent loss in AML (AML-M5). N-CoR displayed significantly higher level of serine specific phosphorylation in almost all AML-M5 derived cells and was subjected to processing by AML-M5 specific aberrant protease activity. To identify the kinase linked to N-CoR phosphorylation, a library of activated kinases was screened with the extracts of AML cells; leading to the identification of Akt as the putative kinase linked to N-CoR phosphorylation. Consistent with this finding, a constitutively active Akt consistently phosphorylated N-CoR leading to its misfolding; while the therapeutic and genetic ablation of Akt largely abrogated the MCDL of N-CoR in AML-M5 cells. Site directed mutagenic analysis of N-CoR identified serine 1450 as the crucial residue whose phosphorylation by Akt was essential for the misfolding and loss of N-CoR protein. Moreover, Akt-induced phosphorylation of N-CoR contributed to the de-repression of Flt3, suggesting a cross talk between Akt signaling and N-CoR misfolding pathway in the pathogenesis of AML-M5. The N-CoR misfolding pathway could be the common downstream thread of pleiotropic Akt signaling activated by various oncogenic insults in some subtypes of leukemia and solid tumors.

Show MeSH
Related in: MedlinePlus