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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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Akt promotes N-CoR misfolding.A, Relative solubility/insolubility of flag tagged N-CoR expressed with the constitutively active Akt (myr-Akt) in 293T cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions were separated by high-speed centrifugation and N-CoR levels in each fraction were determined by western blotting assay using Flag antibody. The constitutively active Akt (myr-Akt) caused a significant increase in insoluble N-CoR protein levels. The relative solubility/insolubility of β-actin in each fraction served as a control. The level of total protein in each fraction was determined by coomassie blue staining. B, Subcellular distribution of N-CoR-Flag (red fluorescence) in 293T cells transfected with flag-tagged N-CoR with or without the constitutively active GFP tagged myr-Akt (green fluorescence) was determined by confocal microscopy. N-CoR-Flag was targeted to the cytosol when co-expressed with GFP-myr-Akt, while N-CoR-Flag expressed with empty vector was localized mainly in the nucleus. C, Inhibition of Akt leads to N-CoR stabilization. N-CoR and Akt level in THP- 1 cells treated with Akt or control siRNA was determined by western blotting with the respective antibodies (left panel). Levels of N-CoR, pAkt and Akt in THP-1 cells treated in a dose dependent manner with Akti-X, the commercially available specific inhibitor of Akt, was similarly determined (right panel). D, Therapeutic inhibition of Akt leads to N-CoR stabilization in primary and secondary AML-M5 cells. Levels of N-CoR, pAkt and Akt in AML-M5 cells (left panel) and human primary AML-M5 cells (right panel) treated in a dose dependent manner with Akti-X was determined. E, Native N-CoR conformation is rescued by Akt abrogation. Relative solubility/insolubility of N-CoR protein in Akt siRNA or Akti-X treated THP-1 cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions of 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. THP-1 cells treated with AEBSF or genistein was used as controls. The relative solubility/insolubility of β-actin in each fraction served as an experimental control. The level of total of protein in each fraction was determined by coomassie blue staining. F, Subcellular distribution of N-CoR (red signal) in THP-1 cells treated with AEBSF at 200 µM, Akt siRNA or Akti-X at 2.5 µM was determined by confocal microscopy.
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pone-0070891-g003: Akt promotes N-CoR misfolding.A, Relative solubility/insolubility of flag tagged N-CoR expressed with the constitutively active Akt (myr-Akt) in 293T cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions were separated by high-speed centrifugation and N-CoR levels in each fraction were determined by western blotting assay using Flag antibody. The constitutively active Akt (myr-Akt) caused a significant increase in insoluble N-CoR protein levels. The relative solubility/insolubility of β-actin in each fraction served as a control. The level of total protein in each fraction was determined by coomassie blue staining. B, Subcellular distribution of N-CoR-Flag (red fluorescence) in 293T cells transfected with flag-tagged N-CoR with or without the constitutively active GFP tagged myr-Akt (green fluorescence) was determined by confocal microscopy. N-CoR-Flag was targeted to the cytosol when co-expressed with GFP-myr-Akt, while N-CoR-Flag expressed with empty vector was localized mainly in the nucleus. C, Inhibition of Akt leads to N-CoR stabilization. N-CoR and Akt level in THP- 1 cells treated with Akt or control siRNA was determined by western blotting with the respective antibodies (left panel). Levels of N-CoR, pAkt and Akt in THP-1 cells treated in a dose dependent manner with Akti-X, the commercially available specific inhibitor of Akt, was similarly determined (right panel). D, Therapeutic inhibition of Akt leads to N-CoR stabilization in primary and secondary AML-M5 cells. Levels of N-CoR, pAkt and Akt in AML-M5 cells (left panel) and human primary AML-M5 cells (right panel) treated in a dose dependent manner with Akti-X was determined. E, Native N-CoR conformation is rescued by Akt abrogation. Relative solubility/insolubility of N-CoR protein in Akt siRNA or Akti-X treated THP-1 cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions of 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. THP-1 cells treated with AEBSF or genistein was used as controls. The relative solubility/insolubility of β-actin in each fraction served as an experimental control. The level of total of protein in each fraction was determined by coomassie blue staining. F, Subcellular distribution of N-CoR (red signal) in THP-1 cells treated with AEBSF at 200 µM, Akt siRNA or Akti-X at 2.5 µM was determined by confocal microscopy.

Mentions: The role of Akt in N-CoR misfolding was further characterized by employing myr-Akt, a constitutively activated form of Akt in which the myristoylation signal is linked in frame to the Akt sequence [44]. The conformation of flag-tagged N-CoR co-expressed with myr-Akt in 293T cells was determined through protein solubility assay and analysis of sub-cellular distribution. When N-CoR was co-expressed with myr-Akt, the level of insoluble N-CoR protein was significantly increased, suggesting a direct role of myr-Akt in N-CoR misfolding (Fig. 3A). Consistent with the findings of the solubility assay, N-CoR co-expressed with myr-Akt displayed a predominantly cytosolic distribution pattern, suggesting that it has adopted a misfolded conformation (Fig. 3B, lower panel). To further define the role of Akt in N-CoR misfolding, the solubility and sub-cellular distribution of N-CoR in THP-1 cells was determined after Akt inhibition with siRNA or Akti-X, a commercially available selective small molecular inhibitor of Akt [36], [37]. Selective Akt inhibition in THP-1 cells by either means led to the stabilization of full length N-CoR (Fig. 3C). As observed in THP-1 cells, selective Akt inhibition with Akti-X also led to N-CoR stabilization in other AML-M5 derived cells (Fig. 3D left panel) as well as in human primary AML-M5 cells (Fig. 3D right panel). Interestingly, N-CoR stabilized by Akt siRNA or Akti-X was detergent soluble (Fig. 3E) and predominantly nuclear (Fig. 3F), suggesting that N-CoR regained its native conformation after Akt inhibition. Taken together, these observations suggested a direct role of Akt in the misfolding of N-CoR protein.


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)

Akt promotes N-CoR misfolding.A, Relative solubility/insolubility of flag tagged N-CoR expressed with the constitutively active Akt (myr-Akt) in 293T cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions were separated by high-speed centrifugation and N-CoR levels in each fraction were determined by western blotting assay using Flag antibody. The constitutively active Akt (myr-Akt) caused a significant increase in insoluble N-CoR protein levels. The relative solubility/insolubility of β-actin in each fraction served as a control. The level of total protein in each fraction was determined by coomassie blue staining. B, Subcellular distribution of N-CoR-Flag (red fluorescence) in 293T cells transfected with flag-tagged N-CoR with or without the constitutively active GFP tagged myr-Akt (green fluorescence) was determined by confocal microscopy. N-CoR-Flag was targeted to the cytosol when co-expressed with GFP-myr-Akt, while N-CoR-Flag expressed with empty vector was localized mainly in the nucleus. C, Inhibition of Akt leads to N-CoR stabilization. N-CoR and Akt level in THP- 1 cells treated with Akt or control siRNA was determined by western blotting with the respective antibodies (left panel). Levels of N-CoR, pAkt and Akt in THP-1 cells treated in a dose dependent manner with Akti-X, the commercially available specific inhibitor of Akt, was similarly determined (right panel). D, Therapeutic inhibition of Akt leads to N-CoR stabilization in primary and secondary AML-M5 cells. Levels of N-CoR, pAkt and Akt in AML-M5 cells (left panel) and human primary AML-M5 cells (right panel) treated in a dose dependent manner with Akti-X was determined. E, Native N-CoR conformation is rescued by Akt abrogation. Relative solubility/insolubility of N-CoR protein in Akt siRNA or Akti-X treated THP-1 cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions of 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. THP-1 cells treated with AEBSF or genistein was used as controls. The relative solubility/insolubility of β-actin in each fraction served as an experimental control. The level of total of protein in each fraction was determined by coomassie blue staining. F, Subcellular distribution of N-CoR (red signal) in THP-1 cells treated with AEBSF at 200 µM, Akt siRNA or Akti-X at 2.5 µM was determined by confocal microscopy.
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getmorefigures.php?uid=PMC3733915&req=5

pone-0070891-g003: Akt promotes N-CoR misfolding.A, Relative solubility/insolubility of flag tagged N-CoR expressed with the constitutively active Akt (myr-Akt) in 293T cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions were separated by high-speed centrifugation and N-CoR levels in each fraction were determined by western blotting assay using Flag antibody. The constitutively active Akt (myr-Akt) caused a significant increase in insoluble N-CoR protein levels. The relative solubility/insolubility of β-actin in each fraction served as a control. The level of total protein in each fraction was determined by coomassie blue staining. B, Subcellular distribution of N-CoR-Flag (red fluorescence) in 293T cells transfected with flag-tagged N-CoR with or without the constitutively active GFP tagged myr-Akt (green fluorescence) was determined by confocal microscopy. N-CoR-Flag was targeted to the cytosol when co-expressed with GFP-myr-Akt, while N-CoR-Flag expressed with empty vector was localized mainly in the nucleus. C, Inhibition of Akt leads to N-CoR stabilization. N-CoR and Akt level in THP- 1 cells treated with Akt or control siRNA was determined by western blotting with the respective antibodies (left panel). Levels of N-CoR, pAkt and Akt in THP-1 cells treated in a dose dependent manner with Akti-X, the commercially available specific inhibitor of Akt, was similarly determined (right panel). D, Therapeutic inhibition of Akt leads to N-CoR stabilization in primary and secondary AML-M5 cells. Levels of N-CoR, pAkt and Akt in AML-M5 cells (left panel) and human primary AML-M5 cells (right panel) treated in a dose dependent manner with Akti-X was determined. E, Native N-CoR conformation is rescued by Akt abrogation. Relative solubility/insolubility of N-CoR protein in Akt siRNA or Akti-X treated THP-1 cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions of 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. THP-1 cells treated with AEBSF or genistein was used as controls. The relative solubility/insolubility of β-actin in each fraction served as an experimental control. The level of total of protein in each fraction was determined by coomassie blue staining. F, Subcellular distribution of N-CoR (red signal) in THP-1 cells treated with AEBSF at 200 µM, Akt siRNA or Akti-X at 2.5 µM was determined by confocal microscopy.
Mentions: The role of Akt in N-CoR misfolding was further characterized by employing myr-Akt, a constitutively activated form of Akt in which the myristoylation signal is linked in frame to the Akt sequence [44]. The conformation of flag-tagged N-CoR co-expressed with myr-Akt in 293T cells was determined through protein solubility assay and analysis of sub-cellular distribution. When N-CoR was co-expressed with myr-Akt, the level of insoluble N-CoR protein was significantly increased, suggesting a direct role of myr-Akt in N-CoR misfolding (Fig. 3A). Consistent with the findings of the solubility assay, N-CoR co-expressed with myr-Akt displayed a predominantly cytosolic distribution pattern, suggesting that it has adopted a misfolded conformation (Fig. 3B, lower panel). To further define the role of Akt in N-CoR misfolding, the solubility and sub-cellular distribution of N-CoR in THP-1 cells was determined after Akt inhibition with siRNA or Akti-X, a commercially available selective small molecular inhibitor of Akt [36], [37]. Selective Akt inhibition in THP-1 cells by either means led to the stabilization of full length N-CoR (Fig. 3C). As observed in THP-1 cells, selective Akt inhibition with Akti-X also led to N-CoR stabilization in other AML-M5 derived cells (Fig. 3D left panel) as well as in human primary AML-M5 cells (Fig. 3D right panel). Interestingly, N-CoR stabilized by Akt siRNA or Akti-X was detergent soluble (Fig. 3E) and predominantly nuclear (Fig. 3F), suggesting that N-CoR regained its native conformation after Akt inhibition. Taken together, these observations suggested a direct role of Akt in the misfolding of N-CoR protein.

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