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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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Serine 1450 phosphorylation is critical for Akt-induced N-CoR misfolding.A, Serine to alanine substitution at 1450 abrogated Akt-induced N-CoR misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450A or T1925A mutants) in 293T cells transfected with the constitutively active myr-Akt 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 with flag antibody (upper panel). The relative solubility/insolubility of β-actin in each fraction was determined as a control (middle panel). The level of total of protein in each fraction was determined by coomassie blue staining (lower panel). B, N-CoR S1450E, the phosphomimetic mutant of N-CoR, displayed signs of misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450E) in 293T cells was determined by protein solubility assay. C, S1450E is localized in cytosol. Subcellular distribution of flag-tagged N-CoR (WT, S1450A and S1450E) was determined by confocal microscopy after staining of the cells transfected with each plasmid with flag antibody. D, S1450E can induce ER stress. Level of GRP78/BiP (green) in 293T cells transfected with S1450E (red) was determined by confocal microscopy after staining the cells with flag and GRP78/BiP antibodies.
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pone-0070891-g006: Serine 1450 phosphorylation is critical for Akt-induced N-CoR misfolding.A, Serine to alanine substitution at 1450 abrogated Akt-induced N-CoR misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450A or T1925A mutants) in 293T cells transfected with the constitutively active myr-Akt 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 with flag antibody (upper panel). The relative solubility/insolubility of β-actin in each fraction was determined as a control (middle panel). The level of total of protein in each fraction was determined by coomassie blue staining (lower panel). B, N-CoR S1450E, the phosphomimetic mutant of N-CoR, displayed signs of misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450E) in 293T cells was determined by protein solubility assay. C, S1450E is localized in cytosol. Subcellular distribution of flag-tagged N-CoR (WT, S1450A and S1450E) was determined by confocal microscopy after staining of the cells transfected with each plasmid with flag antibody. D, S1450E can induce ER stress. Level of GRP78/BiP (green) in 293T cells transfected with S1450E (red) was determined by confocal microscopy after staining the cells with flag and GRP78/BiP antibodies.

Mentions: Next, to investigate the link between serine 1450 phosphorylation and misfolding, the effect of myr-Akt on the misfolding of N-CoR S1450A or N-CoR T1925A was analyzed in 293T cells transfected with myr-Akt and flag tagged N-CoR S1450A or N-CoR T1925A plasmids. While myr-Akt could effectively render the N-CoR T1925A mutant insoluble, the N-CoR S1450A mutant remained largely soluble (Fig. 6A), suggesting a crucial role of serine 1450 in Akt-induced misfolding of N-CoR. Phosphorylation usually triggers a qualitative change in protein conformation by conferring a negative charge to the residue it phosphorylates. Therefore, to further confirm that the Akt-induced N-CoR phosphorylation at serine 1450 actually triggers the misfolding of N-CoR protein, we tested the solubility and subcellular distribution of N-CoR S1450E, a phosphomimic mutant of N-CoR in which the serine (S) at 1450 was replaced with glutamic acid (E) that carries a native negative charge due to its -COOH group (Fig. S5). When expressed in 293T cells, a significant portion of N-CoR S1450E was accumulated in the insoluble fraction and was predominantly localized in the cytosol (Fig. 6B & C), suggesting that N-CoR S1450E mutant could spontaneously assume a misfolded conformation. Like the original misfolded N-CoR, N-CoR S1450E could also induce ER stress (a key impact of misfolded protein) as evidenced by the up regulation of a key ER stress marker GRP78/BiP in cells expressing N-CoR S1450E (Fig. 6D). Collectively, these findings suggested that Akt-induced phosphorylation of N-CoR at serine 1450 triggers a conformational change in N-CoR leading to its misfolding and eventual loss in AML-M5.


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)

Serine 1450 phosphorylation is critical for Akt-induced N-CoR misfolding.A, Serine to alanine substitution at 1450 abrogated Akt-induced N-CoR misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450A or T1925A mutants) in 293T cells transfected with the constitutively active myr-Akt 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 with flag antibody (upper panel). The relative solubility/insolubility of β-actin in each fraction was determined as a control (middle panel). The level of total of protein in each fraction was determined by coomassie blue staining (lower panel). B, N-CoR S1450E, the phosphomimetic mutant of N-CoR, displayed signs of misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450E) in 293T cells was determined by protein solubility assay. C, S1450E is localized in cytosol. Subcellular distribution of flag-tagged N-CoR (WT, S1450A and S1450E) was determined by confocal microscopy after staining of the cells transfected with each plasmid with flag antibody. D, S1450E can induce ER stress. Level of GRP78/BiP (green) in 293T cells transfected with S1450E (red) was determined by confocal microscopy after staining the cells with flag and GRP78/BiP antibodies.
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pone-0070891-g006: Serine 1450 phosphorylation is critical for Akt-induced N-CoR misfolding.A, Serine to alanine substitution at 1450 abrogated Akt-induced N-CoR misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450A or T1925A mutants) in 293T cells transfected with the constitutively active myr-Akt 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 with flag antibody (upper panel). The relative solubility/insolubility of β-actin in each fraction was determined as a control (middle panel). The level of total of protein in each fraction was determined by coomassie blue staining (lower panel). B, N-CoR S1450E, the phosphomimetic mutant of N-CoR, displayed signs of misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450E) in 293T cells was determined by protein solubility assay. C, S1450E is localized in cytosol. Subcellular distribution of flag-tagged N-CoR (WT, S1450A and S1450E) was determined by confocal microscopy after staining of the cells transfected with each plasmid with flag antibody. D, S1450E can induce ER stress. Level of GRP78/BiP (green) in 293T cells transfected with S1450E (red) was determined by confocal microscopy after staining the cells with flag and GRP78/BiP antibodies.
Mentions: Next, to investigate the link between serine 1450 phosphorylation and misfolding, the effect of myr-Akt on the misfolding of N-CoR S1450A or N-CoR T1925A was analyzed in 293T cells transfected with myr-Akt and flag tagged N-CoR S1450A or N-CoR T1925A plasmids. While myr-Akt could effectively render the N-CoR T1925A mutant insoluble, the N-CoR S1450A mutant remained largely soluble (Fig. 6A), suggesting a crucial role of serine 1450 in Akt-induced misfolding of N-CoR. Phosphorylation usually triggers a qualitative change in protein conformation by conferring a negative charge to the residue it phosphorylates. Therefore, to further confirm that the Akt-induced N-CoR phosphorylation at serine 1450 actually triggers the misfolding of N-CoR protein, we tested the solubility and subcellular distribution of N-CoR S1450E, a phosphomimic mutant of N-CoR in which the serine (S) at 1450 was replaced with glutamic acid (E) that carries a native negative charge due to its -COOH group (Fig. S5). When expressed in 293T cells, a significant portion of N-CoR S1450E was accumulated in the insoluble fraction and was predominantly localized in the cytosol (Fig. 6B & C), suggesting that N-CoR S1450E mutant could spontaneously assume a misfolded conformation. Like the original misfolded N-CoR, N-CoR S1450E could also induce ER stress (a key impact of misfolded protein) as evidenced by the up regulation of a key ER stress marker GRP78/BiP in cells expressing N-CoR S1450E (Fig. 6D). Collectively, these findings suggested that Akt-induced phosphorylation of N-CoR at serine 1450 triggers a conformational change in N-CoR leading to its misfolding and eventual loss in AML-M5.

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