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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

Proteasome degradation through SUMOylation has a role in CBY1 reduced stability associated with BCR-ABL1 TK-mediated binding with 14-3-3σ.A- Cytoplasmic CBY1 expression was upraised at 24th hour of treatment with Bortezomib (5nM) in C22orf2 K562 cells; B- Reduction of CBY1 SUMOylation was associated with CBY1 upraised expression in response to compounds (IM, BV02 and RAD001) which impair the interaction with 14-3-3σ. Conversely, CBY1 SUMOylation was increased in response to the JNK inhibitor, which precludes CBY1 release from 14-3-3σ and reduces CBY1 cytoplasmic levels; C- Higher CBY1 SUMOylation was seen in MCF from bone marrow samples of 12 CML patients at diagnosis compared to pooled HD. Clinical details of CML patients included in our study are illustrated in the S1 Table. The two blots shown here have been run separately and confirmed by two additional independent experiments. D- CBY1 SUMOylation in MCF from CML patients was inversely correlated with the expression of CBY1. Signal intensities of WB obtained with equal amounts of protein from CML and HD samples. CBY1 expression exhibited a decrease of more than 50% with respect to the normal control, while SUMOylation showed an almost 1,5-fold increment compared to the normal control (signal intensity of HD protein pool was set to 1 to represent the reference value). See legend to Fig 1 for technical details.
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pone.0131074.g006: Proteasome degradation through SUMOylation has a role in CBY1 reduced stability associated with BCR-ABL1 TK-mediated binding with 14-3-3σ.A- Cytoplasmic CBY1 expression was upraised at 24th hour of treatment with Bortezomib (5nM) in C22orf2 K562 cells; B- Reduction of CBY1 SUMOylation was associated with CBY1 upraised expression in response to compounds (IM, BV02 and RAD001) which impair the interaction with 14-3-3σ. Conversely, CBY1 SUMOylation was increased in response to the JNK inhibitor, which precludes CBY1 release from 14-3-3σ and reduces CBY1 cytoplasmic levels; C- Higher CBY1 SUMOylation was seen in MCF from bone marrow samples of 12 CML patients at diagnosis compared to pooled HD. Clinical details of CML patients included in our study are illustrated in the S1 Table. The two blots shown here have been run separately and confirmed by two additional independent experiments. D- CBY1 SUMOylation in MCF from CML patients was inversely correlated with the expression of CBY1. Signal intensities of WB obtained with equal amounts of protein from CML and HD samples. CBY1 expression exhibited a decrease of more than 50% with respect to the normal control, while SUMOylation showed an almost 1,5-fold increment compared to the normal control (signal intensity of HD protein pool was set to 1 to represent the reference value). See legend to Fig 1 for technical details.

Mentions: Selective degradation of the great majority of intracellular proteins is executed by the ubiquitin proteasome system (UPS) [29–31]. Bortezomib, an N-protected dipeptide, whose boronic acid at the C-terminus binds and inhibits the catalytic site of the 26S proteasome, was used to assess whether enhanced UPS degradation is implicated in CBY1 down-modulation associated with BCR-ABL1 [29]. In C22orf2 K562, CBY1 exhibited a significant increment in response to bortezomib (5 nM for 24 h) (p<.001) (Fig 6A). The findings raise the possibility that UPS is a component of CBY1 down-modulation in CML. The detection of small ubiquitin-like modifier (SUMO) groups in the CBY1 sequence addressed further investigation towards SUMOylation as a mechanism involved in CBY1 increased degradation associated with BCR-ABL1, CBY1 SUMOylation status was investigated under experimental conditions affecting CBY1/14-3-3σ interaction used in the first part of our work. To this purpose, CBY1 IP products from C22orf2 K562 cells treated with drugs either promoting (IM, BV02 and RAD001) or preventing (SP600125) CBY1 release from 14-3-3σ were probed with anti-SUMO antibodies. CBY1 increment following its release from 14-3-3σ in response to IM, BV02 and RAD001 was associated with a significant reduction in the signal intensities with anti-SUMO antibody (p<0.01) (Fig 6B). Conversely, the reduction of CBY1 expression associated with the persistence of CBY1/14-3-3σ upon SP600125 treatment was associated with a significant increment in the band intensities with anti-SUMO antibody (p<0.001) (Fig 6B). The findings suggest that SUMOylation may intervene in the degradation of CBY1 bound with 14-3-3σ.


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)

Proteasome degradation through SUMOylation has a role in CBY1 reduced stability associated with BCR-ABL1 TK-mediated binding with 14-3-3σ.A- Cytoplasmic CBY1 expression was upraised at 24th hour of treatment with Bortezomib (5nM) in C22orf2 K562 cells; B- Reduction of CBY1 SUMOylation was associated with CBY1 upraised expression in response to compounds (IM, BV02 and RAD001) which impair the interaction with 14-3-3σ. Conversely, CBY1 SUMOylation was increased in response to the JNK inhibitor, which precludes CBY1 release from 14-3-3σ and reduces CBY1 cytoplasmic levels; C- Higher CBY1 SUMOylation was seen in MCF from bone marrow samples of 12 CML patients at diagnosis compared to pooled HD. Clinical details of CML patients included in our study are illustrated in the S1 Table. The two blots shown here have been run separately and confirmed by two additional independent experiments. D- CBY1 SUMOylation in MCF from CML patients was inversely correlated with the expression of CBY1. Signal intensities of WB obtained with equal amounts of protein from CML and HD samples. CBY1 expression exhibited a decrease of more than 50% with respect to the normal control, while SUMOylation showed an almost 1,5-fold increment compared to the normal control (signal intensity of HD protein pool was set to 1 to represent the reference value). See legend to Fig 1 for technical details.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131074.g006: Proteasome degradation through SUMOylation has a role in CBY1 reduced stability associated with BCR-ABL1 TK-mediated binding with 14-3-3σ.A- Cytoplasmic CBY1 expression was upraised at 24th hour of treatment with Bortezomib (5nM) in C22orf2 K562 cells; B- Reduction of CBY1 SUMOylation was associated with CBY1 upraised expression in response to compounds (IM, BV02 and RAD001) which impair the interaction with 14-3-3σ. Conversely, CBY1 SUMOylation was increased in response to the JNK inhibitor, which precludes CBY1 release from 14-3-3σ and reduces CBY1 cytoplasmic levels; C- Higher CBY1 SUMOylation was seen in MCF from bone marrow samples of 12 CML patients at diagnosis compared to pooled HD. Clinical details of CML patients included in our study are illustrated in the S1 Table. The two blots shown here have been run separately and confirmed by two additional independent experiments. D- CBY1 SUMOylation in MCF from CML patients was inversely correlated with the expression of CBY1. Signal intensities of WB obtained with equal amounts of protein from CML and HD samples. CBY1 expression exhibited a decrease of more than 50% with respect to the normal control, while SUMOylation showed an almost 1,5-fold increment compared to the normal control (signal intensity of HD protein pool was set to 1 to represent the reference value). See legend to Fig 1 for technical details.
Mentions: Selective degradation of the great majority of intracellular proteins is executed by the ubiquitin proteasome system (UPS) [29–31]. Bortezomib, an N-protected dipeptide, whose boronic acid at the C-terminus binds and inhibits the catalytic site of the 26S proteasome, was used to assess whether enhanced UPS degradation is implicated in CBY1 down-modulation associated with BCR-ABL1 [29]. In C22orf2 K562, CBY1 exhibited a significant increment in response to bortezomib (5 nM for 24 h) (p<.001) (Fig 6A). The findings raise the possibility that UPS is a component of CBY1 down-modulation in CML. The detection of small ubiquitin-like modifier (SUMO) groups in the CBY1 sequence addressed further investigation towards SUMOylation as a mechanism involved in CBY1 increased degradation associated with BCR-ABL1, CBY1 SUMOylation status was investigated under experimental conditions affecting CBY1/14-3-3σ interaction used in the first part of our work. To this purpose, CBY1 IP products from C22orf2 K562 cells treated with drugs either promoting (IM, BV02 and RAD001) or preventing (SP600125) CBY1 release from 14-3-3σ were probed with anti-SUMO antibodies. CBY1 increment following its release from 14-3-3σ in response to IM, BV02 and RAD001 was associated with a significant reduction in the signal intensities with anti-SUMO antibody (p<0.01) (Fig 6B). Conversely, the reduction of CBY1 expression associated with the persistence of CBY1/14-3-3σ upon SP600125 treatment was associated with a significant increment in the band intensities with anti-SUMO antibody (p<0.001) (Fig 6B). The findings suggest that SUMOylation may intervene in the degradation of CBY1 bound with 14-3-3σ.

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