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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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Misfolded conformation dependent loss (MCDL) of N-CoR in AML-M5.A, An aliquot of whole cell extract of various AML derived cells as mentioned on the top of each lane was resolved in SDS-PAGE and stained with N-CoR antibody. B, An aliquot of whole cell extract of AML-M5 patient samples was resolved in SDS-PAGE and stained with N-CoR antibody. N-CoR level in HL-60 was used as positive control. C, N-CoR is stabilized by AEBSF and genistein. Level of full length and cleaved N-CoR protein in THP-1 cells treated with AEBSF or genistein in a dose dependent manner was determined by western blotting assay using N-CoR antibody. D, Native N-CoR conformation is rescued by genistein but not by AEBSF. Relative solubility/insolubility of N-CoR protein in AEBSF or genistein treated THP-1 cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions of AEBSF- or genistein-treated THP-1 cells were separated by high speed centrifugation and N-CoR level in each fraction was determined by western blotting assay using N-CoR antibody. A HMW (high molecular weight) variant of N-CoR protein, which was part of the insoluble fraction, was detected only in AEBSF treated cells. The relative solubility/insolubility of β-actin in each fraction was used as a control. The level of total of protein in each fraction was determined by coomassie blue staining. E, Subcellular distribution of N-CoR (red signal) in THP-1 cells treated with AEBSF (200 µM) or genistein (50 µM) was determined by confocal microscopy. DNA was stained with DAPI (blue signal). F, Level of serine/threonine phosphorylated N-CoR (upper panel) or total N-CoR (lower panel) in THP-1 cells treated with AEBSF or genistein was determined by staining the immunoprecipitated (IP) N-CoR with a generic phospho serine/threonine antibody or N-CoR antibody respectively. N-CoR protein level in each treated sample used in IP assay was determined by western blotting (right panel).
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pone-0070891-g001: Misfolded conformation dependent loss (MCDL) of N-CoR in AML-M5.A, An aliquot of whole cell extract of various AML derived cells as mentioned on the top of each lane was resolved in SDS-PAGE and stained with N-CoR antibody. B, An aliquot of whole cell extract of AML-M5 patient samples was resolved in SDS-PAGE and stained with N-CoR antibody. N-CoR level in HL-60 was used as positive control. C, N-CoR is stabilized by AEBSF and genistein. Level of full length and cleaved N-CoR protein in THP-1 cells treated with AEBSF or genistein in a dose dependent manner was determined by western blotting assay using N-CoR antibody. D, Native N-CoR conformation is rescued by genistein but not by AEBSF. Relative solubility/insolubility of N-CoR protein in AEBSF or genistein treated THP-1 cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions of AEBSF- or genistein-treated THP-1 cells were separated by high speed centrifugation and N-CoR level in each fraction was determined by western blotting assay using N-CoR antibody. A HMW (high molecular weight) variant of N-CoR protein, which was part of the insoluble fraction, was detected only in AEBSF treated cells. The relative solubility/insolubility of β-actin in each fraction was used as a control. The level of total of protein in each fraction was determined by coomassie blue staining. E, Subcellular distribution of N-CoR (red signal) in THP-1 cells treated with AEBSF (200 µM) or genistein (50 µM) was determined by confocal microscopy. DNA was stained with DAPI (blue signal). F, Level of serine/threonine phosphorylated N-CoR (upper panel) or total N-CoR (lower panel) in THP-1 cells treated with AEBSF or genistein was determined by staining the immunoprecipitated (IP) N-CoR with a generic phospho serine/threonine antibody or N-CoR antibody respectively. N-CoR protein level in each treated sample used in IP assay was determined by western blotting (right panel).

Mentions: Given the key role of N-CoR in the transcriptional repression of Flt3[11], we hypothesized that de-regulation of N-CoR mediated transcriptional control as a result of its misfolding might have a significant implication in the pathogenesis of AML. Therefore, to characterize the misfolding status of N-CoR in various AML subtypes, we first determined the level of natively folded or stable (full length) N-CoR in various AML and non-AML cells through western blotting assay. In contrast to natively folded N-CoR which migrates as a 270 kDa band in western blotting assay, the misfolded N-CoR is evidently unstable and usually appears as a cleaved 100 kDa fragment in the same assay [11]–[14]. When analyzed by western blotting assay, no intact (270 kDa) N-CoR was detected in various AML-M5 derived cell lines; though an intact N-CoR of 270 kDa was evidently presented in all three non-AML-M5 derived cell lines U937, K562 and HL-60 used as controls (Fig. 1A). Instead of full length N-CoR, all AML-M5 derived cell lines contained a cleaved N-CoR fragment of 100 kDa (Fig. 1A). The absence or loss of full length N-CoR in AML-M5 cells was not due to a lack of expression of N-CoR mRNA, as the level of N-CoR transcript in all five AML-M5 derived cells was more or less comparable to that of non-AML-M5 derived cell lines (Fig. S1). Interestingly, N-CoR displayed similar pattern of processing in multiple primary human AML-M5 patient samples, suggesting that N-CoR processing was clinically relevant and not an artifact of cell line system (Fig. 1B).


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)

Misfolded conformation dependent loss (MCDL) of N-CoR in AML-M5.A, An aliquot of whole cell extract of various AML derived cells as mentioned on the top of each lane was resolved in SDS-PAGE and stained with N-CoR antibody. B, An aliquot of whole cell extract of AML-M5 patient samples was resolved in SDS-PAGE and stained with N-CoR antibody. N-CoR level in HL-60 was used as positive control. C, N-CoR is stabilized by AEBSF and genistein. Level of full length and cleaved N-CoR protein in THP-1 cells treated with AEBSF or genistein in a dose dependent manner was determined by western blotting assay using N-CoR antibody. D, Native N-CoR conformation is rescued by genistein but not by AEBSF. Relative solubility/insolubility of N-CoR protein in AEBSF or genistein treated THP-1 cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions of AEBSF- or genistein-treated THP-1 cells were separated by high speed centrifugation and N-CoR level in each fraction was determined by western blotting assay using N-CoR antibody. A HMW (high molecular weight) variant of N-CoR protein, which was part of the insoluble fraction, was detected only in AEBSF treated cells. The relative solubility/insolubility of β-actin in each fraction was used as a control. The level of total of protein in each fraction was determined by coomassie blue staining. E, Subcellular distribution of N-CoR (red signal) in THP-1 cells treated with AEBSF (200 µM) or genistein (50 µM) was determined by confocal microscopy. DNA was stained with DAPI (blue signal). F, Level of serine/threonine phosphorylated N-CoR (upper panel) or total N-CoR (lower panel) in THP-1 cells treated with AEBSF or genistein was determined by staining the immunoprecipitated (IP) N-CoR with a generic phospho serine/threonine antibody or N-CoR antibody respectively. N-CoR protein level in each treated sample used in IP assay was determined by western blotting (right panel).
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pone-0070891-g001: Misfolded conformation dependent loss (MCDL) of N-CoR in AML-M5.A, An aliquot of whole cell extract of various AML derived cells as mentioned on the top of each lane was resolved in SDS-PAGE and stained with N-CoR antibody. B, An aliquot of whole cell extract of AML-M5 patient samples was resolved in SDS-PAGE and stained with N-CoR antibody. N-CoR level in HL-60 was used as positive control. C, N-CoR is stabilized by AEBSF and genistein. Level of full length and cleaved N-CoR protein in THP-1 cells treated with AEBSF or genistein in a dose dependent manner was determined by western blotting assay using N-CoR antibody. D, Native N-CoR conformation is rescued by genistein but not by AEBSF. Relative solubility/insolubility of N-CoR protein in AEBSF or genistein treated THP-1 cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions of AEBSF- or genistein-treated THP-1 cells were separated by high speed centrifugation and N-CoR level in each fraction was determined by western blotting assay using N-CoR antibody. A HMW (high molecular weight) variant of N-CoR protein, which was part of the insoluble fraction, was detected only in AEBSF treated cells. The relative solubility/insolubility of β-actin in each fraction was used as a control. The level of total of protein in each fraction was determined by coomassie blue staining. E, Subcellular distribution of N-CoR (red signal) in THP-1 cells treated with AEBSF (200 µM) or genistein (50 µM) was determined by confocal microscopy. DNA was stained with DAPI (blue signal). F, Level of serine/threonine phosphorylated N-CoR (upper panel) or total N-CoR (lower panel) in THP-1 cells treated with AEBSF or genistein was determined by staining the immunoprecipitated (IP) N-CoR with a generic phospho serine/threonine antibody or N-CoR antibody respectively. N-CoR protein level in each treated sample used in IP assay was determined by western blotting (right panel).
Mentions: Given the key role of N-CoR in the transcriptional repression of Flt3[11], we hypothesized that de-regulation of N-CoR mediated transcriptional control as a result of its misfolding might have a significant implication in the pathogenesis of AML. Therefore, to characterize the misfolding status of N-CoR in various AML subtypes, we first determined the level of natively folded or stable (full length) N-CoR in various AML and non-AML cells through western blotting assay. In contrast to natively folded N-CoR which migrates as a 270 kDa band in western blotting assay, the misfolded N-CoR is evidently unstable and usually appears as a cleaved 100 kDa fragment in the same assay [11]–[14]. When analyzed by western blotting assay, no intact (270 kDa) N-CoR was detected in various AML-M5 derived cell lines; though an intact N-CoR of 270 kDa was evidently presented in all three non-AML-M5 derived cell lines U937, K562 and HL-60 used as controls (Fig. 1A). Instead of full length N-CoR, all AML-M5 derived cell lines contained a cleaved N-CoR fragment of 100 kDa (Fig. 1A). The absence or loss of full length N-CoR in AML-M5 cells was not due to a lack of expression of N-CoR mRNA, as the level of N-CoR transcript in all five AML-M5 derived cells was more or less comparable to that of non-AML-M5 derived cell lines (Fig. S1). Interestingly, N-CoR displayed similar pattern of processing in multiple primary human AML-M5 patient samples, suggesting that N-CoR processing was clinically relevant and not an artifact of cell line system (Fig. 1B).

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