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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

JNK and 14-3-3σ de-phosphorylation at threonine 183 and serine 186, respectively, in response to JNK inhibitor SP600125 prevents the dissolution of CBY1/14-3-3σ complex and leaves steady the cytoplasmic expression of CBY1.A- SP600125 inhibitory effects on its targets (JNK and 14-3-3); B- CBY1 expression and interaction with 14-3-3σ and βcatenin expression and interaction with 14-3-3σ were assayed in the cytoplasmic and nuclear compartments of C22orf2 K562 cells 24th hour of exposure to SP600125. The absence of off-target effects was assayed (Supplementary section, S2 Fig). See legend to Fig 1 for technical details.
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pone.0131074.g004: JNK and 14-3-3σ de-phosphorylation at threonine 183 and serine 186, respectively, in response to JNK inhibitor SP600125 prevents the dissolution of CBY1/14-3-3σ complex and leaves steady the cytoplasmic expression of CBY1.A- SP600125 inhibitory effects on its targets (JNK and 14-3-3); B- CBY1 expression and interaction with 14-3-3σ and βcatenin expression and interaction with 14-3-3σ were assayed in the cytoplasmic and nuclear compartments of C22orf2 K562 cells 24th hour of exposure to SP600125. The absence of off-target effects was assayed (Supplementary section, S2 Fig). See legend to Fig 1 for technical details.

Mentions: The binding of 14-3-3 with client proteins is further regulated by their own post-translational modifications. In particular, phosphorylation of 14-3-3σ and ξ at discrete sites (serine 186 and 184, respectively) by c-Jun N-terminal kinase (JNK) promotes the dissociation of client proteins [26]. The impact of JNK inhibitor SP600125 (25 μM for 24 h) on JNK auto-phosphorylation at threonine 183 and 14-3-3σ phosphorylation at serine 186 was preliminarily evaluated (Fig 4A). Moreover, SP600125 off-target effects on other signaling components critical for interactions of CBY1 and βcatenin with 14-3-3σ were excluded (S3 Fig). The inhibition of JNK-induced phosphorylation of 14-3-3σ in response to SP6000125 significantly enhanced CBY1/14-3-3σ interaction in cytoplasmic compartment of C22orf2 K562 (<0.01) and left steady CBY1/14-3-3σ binding in the nuclear compartment (p<0.1) (Fig 4B). Further investigation is required to elucidate the causes of such difference. In all instance, persistent CBY1 interaction with 14-3-3σ was associated with a significant reduction in CBY1 expression in both compartments of C22orf2 K562 cells (p<0.01) (Fig 4B). These findings support that JNK de-phosphorylation prevents CBY1 dissociation from 14-3-3σ by hindering 14-3-3σ post-translational events critical for client protein release. Persistent CBY1/14-3-3σ interaction was associated with cytoplasmic and nuclear CBY1 reduction, further supporting the hypothesis raised by previous results that the interaction with 14-3-3 impacts CBY1 stability. Conversely, βcatenin/14-3-3σ interaction in the cytoplasmic and nuclear compartment of C22orf2 K562 cells was impaired by SP600125 (p<0.05 and p<0.01, respectively) (Fig 4C). These findings suggest that JNK/14-3-3σ axis is not critical to keep βcatenin bound with 14-3-3σ and that JNK might rather induce βcatenin post-transcriptional modifications at critical residues for binding with 14-3-3σ [27,28]. Moreover, βcatenin expression was significantly reduced in both compartments in response to SP600125 (p< 0.01), further supporting that the release from 14-3-3σ directs cytoplasmic βcatenin towards degradation, concurrently precluding βcatenin nuclear import even in the presence of activated BCR-ABL1 TK. CBY1 increment in response to IM, BV02 and RAD001 and CBY1 absence upon treatment with SP600125 were confirmed in the cytoplasm of parental K562 cell line (S4 Fig).


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)

JNK and 14-3-3σ de-phosphorylation at threonine 183 and serine 186, respectively, in response to JNK inhibitor SP600125 prevents the dissolution of CBY1/14-3-3σ complex and leaves steady the cytoplasmic expression of CBY1.A- SP600125 inhibitory effects on its targets (JNK and 14-3-3); B- CBY1 expression and interaction with 14-3-3σ and βcatenin expression and interaction with 14-3-3σ were assayed in the cytoplasmic and nuclear compartments of C22orf2 K562 cells 24th hour of exposure to SP600125. The absence of off-target effects was assayed (Supplementary section, S2 Fig). See legend to Fig 1 for technical details.
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Related In: Results  -  Collection

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pone.0131074.g004: JNK and 14-3-3σ de-phosphorylation at threonine 183 and serine 186, respectively, in response to JNK inhibitor SP600125 prevents the dissolution of CBY1/14-3-3σ complex and leaves steady the cytoplasmic expression of CBY1.A- SP600125 inhibitory effects on its targets (JNK and 14-3-3); B- CBY1 expression and interaction with 14-3-3σ and βcatenin expression and interaction with 14-3-3σ were assayed in the cytoplasmic and nuclear compartments of C22orf2 K562 cells 24th hour of exposure to SP600125. The absence of off-target effects was assayed (Supplementary section, S2 Fig). See legend to Fig 1 for technical details.
Mentions: The binding of 14-3-3 with client proteins is further regulated by their own post-translational modifications. In particular, phosphorylation of 14-3-3σ and ξ at discrete sites (serine 186 and 184, respectively) by c-Jun N-terminal kinase (JNK) promotes the dissociation of client proteins [26]. The impact of JNK inhibitor SP600125 (25 μM for 24 h) on JNK auto-phosphorylation at threonine 183 and 14-3-3σ phosphorylation at serine 186 was preliminarily evaluated (Fig 4A). Moreover, SP600125 off-target effects on other signaling components critical for interactions of CBY1 and βcatenin with 14-3-3σ were excluded (S3 Fig). The inhibition of JNK-induced phosphorylation of 14-3-3σ in response to SP6000125 significantly enhanced CBY1/14-3-3σ interaction in cytoplasmic compartment of C22orf2 K562 (<0.01) and left steady CBY1/14-3-3σ binding in the nuclear compartment (p<0.1) (Fig 4B). Further investigation is required to elucidate the causes of such difference. In all instance, persistent CBY1 interaction with 14-3-3σ was associated with a significant reduction in CBY1 expression in both compartments of C22orf2 K562 cells (p<0.01) (Fig 4B). These findings support that JNK de-phosphorylation prevents CBY1 dissociation from 14-3-3σ by hindering 14-3-3σ post-translational events critical for client protein release. Persistent CBY1/14-3-3σ interaction was associated with cytoplasmic and nuclear CBY1 reduction, further supporting the hypothesis raised by previous results that the interaction with 14-3-3 impacts CBY1 stability. Conversely, βcatenin/14-3-3σ interaction in the cytoplasmic and nuclear compartment of C22orf2 K562 cells was impaired by SP600125 (p<0.05 and p<0.01, respectively) (Fig 4C). These findings suggest that JNK/14-3-3σ axis is not critical to keep βcatenin bound with 14-3-3σ and that JNK might rather induce βcatenin post-transcriptional modifications at critical residues for binding with 14-3-3σ [27,28]. Moreover, βcatenin expression was significantly reduced in both compartments in response to SP600125 (p< 0.01), further supporting that the release from 14-3-3σ directs cytoplasmic βcatenin towards degradation, concurrently precluding βcatenin nuclear import even in the presence of activated BCR-ABL1 TK. CBY1 increment in response to IM, BV02 and RAD001 and CBY1 absence upon treatment with SP600125 were confirmed in the cytoplasm of parental K562 cell line (S4 Fig).

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