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14-3-3 Binding and Sumoylation Concur to the Down-Modulation of β-catenin Antagonist chibby 1 in Chronic Myeloid Leukemia.

Mancini M, Leo E, Takemaru K, Campi V, Castagnetti F, Soverini S, De Benedittis C, Rosti G, Cavo M, Santucci MA, Martinelli G - PLoS ONE (2015)

Bottom Line: Here we demonstrate that it also arises from reduced protein stability upon binding to 14-3-3σ adapter protein.The ubiquitin proteasome system UPS participates in reducing stability of CBY1 bound with 14-3-3σ through enhanced SUMOylation.Our results open new routes towards the research on molecular pathways promoting the proliferative advantage of leukemic hematopoiesis over the normal counterpart.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Diagnostic and Specialty Medicine-DIMES-Institute of Hematology "L. and A. Seràgnoli". University of Bologna-Medical School, Bologna, Italy.

ABSTRACT
The down-modulation of the β-catenin antagonist Chibby 1 (CBY1) associated with the BCR-ABL1 fusion gene of chronic myeloid leukemia (CML) contributes to the aberrant activation of β-catenin, particularly in leukemic stem cells (LSC) resistant to tyrosine kinase (TK) inhibitors. It is, at least partly, driven by transcriptional events and gene promoter hyper-methylation. Here we demonstrate that it also arises from reduced protein stability upon binding to 14-3-3σ adapter protein. CBY1/14-3-3σ interaction in BCR-ABL1+ cells is mediated by the fusion protein TK and AKT phosphorylation of CBY1 at critical serine 20, and encompasses the 14-3-3σ binding modes I and II involved in the binding with client proteins. Moreover, it is impaired by c-Jun N-terminal kinase (JNK) phosphorylation of 14-3-3σ at serine 186, which promotes dissociation of client proteins. The ubiquitin proteasome system UPS participates in reducing stability of CBY1 bound with 14-3-3σ through enhanced SUMOylation. Our results open new routes towards the research on molecular pathways promoting the proliferative advantage of leukemic hematopoiesis over the normal counterpart.

No MeSH data available.


Related in: MedlinePlus

IM promotes changes in CBY1 and β-catenin expression associated with the dissolution of CBY1 and β-catenin binding with 14-3-3σ. A- Cytoplasmic and nuclear protein analysis was performed after 4 and 24 hours of exposure to IM. In first instance, BCR-ABL1 de-phosphorylation at the critical residue for constitutive activation of the fusion protein enzymatic activity (tytosine-Y-245) was assessed, hence proving IM inhibitory effect on its target at the time other protein expression and interactions were investigated; B- WB and IP/IB have been performed according to published procedures and confirmed in three independent experiments. Densitometric analysis of signal intensities shows a statistically significance difference (p<0.05; Student’s t test) in treated vs untreated cells. Actin and histone H1 were used as control for loading of cytoplasmic and nuclear proteins, respectively. Lack of IM off target effects is shown in the Supplementary section, S2 Fig
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pone.0131074.g001: IM promotes changes in CBY1 and β-catenin expression associated with the dissolution of CBY1 and β-catenin binding with 14-3-3σ. A- Cytoplasmic and nuclear protein analysis was performed after 4 and 24 hours of exposure to IM. In first instance, BCR-ABL1 de-phosphorylation at the critical residue for constitutive activation of the fusion protein enzymatic activity (tytosine-Y-245) was assessed, hence proving IM inhibitory effect on its target at the time other protein expression and interactions were investigated; B- WB and IP/IB have been performed according to published procedures and confirmed in three independent experiments. Densitometric analysis of signal intensities shows a statistically significance difference (p<0.05; Student’s t test) in treated vs untreated cells. Actin and histone H1 were used as control for loading of cytoplasmic and nuclear proteins, respectively. Lack of IM off target effects is shown in the Supplementary section, S2 Fig

Mentions: Our previous study established that CBY1 down-modulation participates in β-catenin activation in CML [15]. CBY1 reduction is contingent upon the BCR-ABL1 TK activity and driven by transcriptional events encompassing DNA hyper-methylation at the promoter-associated CpG islands of the CBY1-encoding gene C22orf2 [18]. Notably, the greater reduction of CBY1 protein compared to transcript suggests that enhanced protein degradation contributes to CBY1 down-modulation in CML hematopoietic progenitors [15]. Previous studies underscored that CBY1 has a central role in β-catenin nuclear export, contingent upon its binding with 14-3-3σ and ξ scaffolding proteins in a stable and tripartite complex encompassing β-catenin [16,17]. Here we investigated the impact of 14-3-3σ binding on CBY1 expression and stability in a BCR-ABL1+ cell context. The study was conducted in parental K562, a BCR-ABL1+ cell line, which exhibits low CBY1 transcript and undetectable protein levels, and in a K562 polyclonal cell population stably transfected with a C22orf2 construct coding for the wt CBY1 (C22orf2 K562) [19]. Due to the inherent lack of CBY1 in parental K562 cell line, most results shown here concern C22orf2 K562, where CBY1 is over-expressed [21]. In first instance, 14-3-3σ IP products were probed with anti-CBY1 or anti-β-catenin antibody and compared for signal intensities under experimental conditions hampering their interaction with the scaffolding protein. The choice of performing IP with anti-14-3-3σ antibody was dictated by the absence of significant differences in 14-3-3σ levels in treated cells compared to untreated controls (see S1 Fig). Our previous studies suggested that reduced CBY1 expression is contingent upon the BCR-ABL1 TK activity. A significant increase in both cytoplasmic and nuclear CBY1 levels was, in fact, seen both in parental and C22orf2 K562 cell lines after 4 and 24 h of exposure to IM (2 μM) (p<0.05) (Fig 1A). CBY1 induction in response to IM was, at least partly, driven by enhanced transcription following gene promoter de-methylation (S2 Fig) [18]. It clearly correlated with the nuclear export of β-catenin, which is followed by β-catenin degradation and inactivation in the cytoplasm (Fig 1A) [9]. Further investigation established that 14-3-3σ binding has a role in changes of CBY1 and β-catenin expression and sub-cellular partitioning in response to IM. BCR-ABL1 TK inactivation after 4 and 24 h of exposure to IM was, in fact, associated with a significant reduction of CBY1 and βcatenin interaction with 14-3-3σ in cytoplasmic and nuclear compartments of C22orf2 K562 (p<0.05) (Fig 1A and 1B). Unlike CBY1, βcatenin release from 14-3-3σ in response to IM was associated with an initial significant increase (4h) in the cytoplasmic compartment, due to its nuclear export (p<0.05), followed by a complete loss, due to its degradation (Fig 1A) [9]. These findings suggest that CBY1 and βcatenin dissociation from 14-3-3σ in response to IM has opposite effects on CBY1 and βcatenin expression.


14-3-3 Binding and Sumoylation Concur to the Down-Modulation of β-catenin Antagonist chibby 1 in Chronic Myeloid Leukemia.

Mancini M, Leo E, Takemaru K, Campi V, Castagnetti F, Soverini S, De Benedittis C, Rosti G, Cavo M, Santucci MA, Martinelli G - PLoS ONE (2015)

IM promotes changes in CBY1 and β-catenin expression associated with the dissolution of CBY1 and β-catenin binding with 14-3-3σ. A- Cytoplasmic and nuclear protein analysis was performed after 4 and 24 hours of exposure to IM. In first instance, BCR-ABL1 de-phosphorylation at the critical residue for constitutive activation of the fusion protein enzymatic activity (tytosine-Y-245) was assessed, hence proving IM inhibitory effect on its target at the time other protein expression and interactions were investigated; B- WB and IP/IB have been performed according to published procedures and confirmed in three independent experiments. Densitometric analysis of signal intensities shows a statistically significance difference (p<0.05; Student’s t test) in treated vs untreated cells. Actin and histone H1 were used as control for loading of cytoplasmic and nuclear proteins, respectively. Lack of IM off target effects is shown in the Supplementary section, S2 Fig
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492953&req=5

pone.0131074.g001: IM promotes changes in CBY1 and β-catenin expression associated with the dissolution of CBY1 and β-catenin binding with 14-3-3σ. A- Cytoplasmic and nuclear protein analysis was performed after 4 and 24 hours of exposure to IM. In first instance, BCR-ABL1 de-phosphorylation at the critical residue for constitutive activation of the fusion protein enzymatic activity (tytosine-Y-245) was assessed, hence proving IM inhibitory effect on its target at the time other protein expression and interactions were investigated; B- WB and IP/IB have been performed according to published procedures and confirmed in three independent experiments. Densitometric analysis of signal intensities shows a statistically significance difference (p<0.05; Student’s t test) in treated vs untreated cells. Actin and histone H1 were used as control for loading of cytoplasmic and nuclear proteins, respectively. Lack of IM off target effects is shown in the Supplementary section, S2 Fig
Mentions: Our previous study established that CBY1 down-modulation participates in β-catenin activation in CML [15]. CBY1 reduction is contingent upon the BCR-ABL1 TK activity and driven by transcriptional events encompassing DNA hyper-methylation at the promoter-associated CpG islands of the CBY1-encoding gene C22orf2 [18]. Notably, the greater reduction of CBY1 protein compared to transcript suggests that enhanced protein degradation contributes to CBY1 down-modulation in CML hematopoietic progenitors [15]. Previous studies underscored that CBY1 has a central role in β-catenin nuclear export, contingent upon its binding with 14-3-3σ and ξ scaffolding proteins in a stable and tripartite complex encompassing β-catenin [16,17]. Here we investigated the impact of 14-3-3σ binding on CBY1 expression and stability in a BCR-ABL1+ cell context. The study was conducted in parental K562, a BCR-ABL1+ cell line, which exhibits low CBY1 transcript and undetectable protein levels, and in a K562 polyclonal cell population stably transfected with a C22orf2 construct coding for the wt CBY1 (C22orf2 K562) [19]. Due to the inherent lack of CBY1 in parental K562 cell line, most results shown here concern C22orf2 K562, where CBY1 is over-expressed [21]. In first instance, 14-3-3σ IP products were probed with anti-CBY1 or anti-β-catenin antibody and compared for signal intensities under experimental conditions hampering their interaction with the scaffolding protein. The choice of performing IP with anti-14-3-3σ antibody was dictated by the absence of significant differences in 14-3-3σ levels in treated cells compared to untreated controls (see S1 Fig). Our previous studies suggested that reduced CBY1 expression is contingent upon the BCR-ABL1 TK activity. A significant increase in both cytoplasmic and nuclear CBY1 levels was, in fact, seen both in parental and C22orf2 K562 cell lines after 4 and 24 h of exposure to IM (2 μM) (p<0.05) (Fig 1A). CBY1 induction in response to IM was, at least partly, driven by enhanced transcription following gene promoter de-methylation (S2 Fig) [18]. It clearly correlated with the nuclear export of β-catenin, which is followed by β-catenin degradation and inactivation in the cytoplasm (Fig 1A) [9]. Further investigation established that 14-3-3σ binding has a role in changes of CBY1 and β-catenin expression and sub-cellular partitioning in response to IM. BCR-ABL1 TK inactivation after 4 and 24 h of exposure to IM was, in fact, associated with a significant reduction of CBY1 and βcatenin interaction with 14-3-3σ in cytoplasmic and nuclear compartments of C22orf2 K562 (p<0.05) (Fig 1A and 1B). Unlike CBY1, βcatenin release from 14-3-3σ in response to IM was associated with an initial significant increase (4h) in the cytoplasmic compartment, due to its nuclear export (p<0.05), followed by a complete loss, due to its degradation (Fig 1A) [9]. These findings suggest that CBY1 and βcatenin dissociation from 14-3-3σ in response to IM has opposite effects on CBY1 and βcatenin expression.

Bottom Line: Here we demonstrate that it also arises from reduced protein stability upon binding to 14-3-3σ adapter protein.The ubiquitin proteasome system UPS participates in reducing stability of CBY1 bound with 14-3-3σ through enhanced SUMOylation.Our results open new routes towards the research on molecular pathways promoting the proliferative advantage of leukemic hematopoiesis over the normal counterpart.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Diagnostic and Specialty Medicine-DIMES-Institute of Hematology "L. and A. Seràgnoli". University of Bologna-Medical School, Bologna, Italy.

ABSTRACT
The down-modulation of the β-catenin antagonist Chibby 1 (CBY1) associated with the BCR-ABL1 fusion gene of chronic myeloid leukemia (CML) contributes to the aberrant activation of β-catenin, particularly in leukemic stem cells (LSC) resistant to tyrosine kinase (TK) inhibitors. It is, at least partly, driven by transcriptional events and gene promoter hyper-methylation. Here we demonstrate that it also arises from reduced protein stability upon binding to 14-3-3σ adapter protein. CBY1/14-3-3σ interaction in BCR-ABL1+ cells is mediated by the fusion protein TK and AKT phosphorylation of CBY1 at critical serine 20, and encompasses the 14-3-3σ binding modes I and II involved in the binding with client proteins. Moreover, it is impaired by c-Jun N-terminal kinase (JNK) phosphorylation of 14-3-3σ at serine 186, which promotes dissociation of client proteins. The ubiquitin proteasome system UPS participates in reducing stability of CBY1 bound with 14-3-3σ through enhanced SUMOylation. Our results open new routes towards the research on molecular pathways promoting the proliferative advantage of leukemic hematopoiesis over the normal counterpart.

No MeSH data available.


Related in: MedlinePlus