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Functional characterization of a BCL10 isoform in the rainbow trout Oncorhynchus mykiss.

Mazzone P, Scudiero I, Coccia E, Ferravante A, Paolucci M, D'Andrea EL, Varricchio E, Pizzulo M, Reale C, Zotti T, Vito P, Stilo R - FEBS Open Bio (2015)

Bottom Line: Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20.Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein.This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

View Article: PubMed Central - PubMed

Affiliation: Biogem, Via Camporeale, Ariano Irpino (AV), Italy.

ABSTRACT
The complexes formed by BCL10, MALT1 and members of the family of CARMA proteins have recently been the focus of much attention because they represent a key mechanism for regulating activation of the transcription factor NF-κB. Here, we report the functional characterization of a novel isoform of BCL10 in the trout Oncorhynchus mykiss, which we named tBCL10. tBCL10 dimerizes, binds to components of the CBM complex and forms cytoplasmic filaments. Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20. Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein. This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

No MeSH data available.


Related in: MedlinePlus

tBCL10 expression. (A–B) Immunoblot analysis of lysates from HEK293 cells transfected with the indicated expression vectors. Were indicated, prior to SDS–PAGE separation cell lysates were treated with 10 units of calf intestinal phosphatase (CIP) for 30 min at 37 °C. (C) Immunoblot analysis of proteic extracts from rainbow trout organs probed with anti-hBCL10. Lysates from HEK293T cells transfected with tBCL10 were used as a positive control (arrow).
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f0010: tBCL10 expression. (A–B) Immunoblot analysis of lysates from HEK293 cells transfected with the indicated expression vectors. Were indicated, prior to SDS–PAGE separation cell lysates were treated with 10 units of calf intestinal phosphatase (CIP) for 30 min at 37 °C. (C) Immunoblot analysis of proteic extracts from rainbow trout organs probed with anti-hBCL10. Lysates from HEK293T cells transfected with tBCL10 were used as a positive control (arrow).

Mentions: When analyzed in immunoblot assay, tBCL10 expressed in mammalian cells migrates as a 28 kDa protein (Fig. 2A). Interestingly, a rabbit antisera raised against hBCL10 also recognizes tBCL10 (Fig. 2A, right panel). In these expression experiments, we noticed that while hBCL10 migrates as a doublet on SDS–PAGE due to phosphorylation of the protein [25,26], tBCL10 occurs as a single band, suggesting that tBCL10 is not target of phosphorylation events. Indeed, experiments in which lysates were treated with phosphatase prior to immunoblot analysis confirm this possibility (Fig. 2B). Finally, an immunoblot assay carried out on proteic lysates extracted from various rainbow trout organs and tissues indicates that a band corresponding to the molecular weight of tBCL10 is expressed in spleen and, less intensely, in kidney (Fig. 2C). Additional bands with higher molecular weight are detectable, possibly corresponding to other BCL10 isoforms encoded by the genome of O.mykiss.


Functional characterization of a BCL10 isoform in the rainbow trout Oncorhynchus mykiss.

Mazzone P, Scudiero I, Coccia E, Ferravante A, Paolucci M, D'Andrea EL, Varricchio E, Pizzulo M, Reale C, Zotti T, Vito P, Stilo R - FEBS Open Bio (2015)

tBCL10 expression. (A–B) Immunoblot analysis of lysates from HEK293 cells transfected with the indicated expression vectors. Were indicated, prior to SDS–PAGE separation cell lysates were treated with 10 units of calf intestinal phosphatase (CIP) for 30 min at 37 °C. (C) Immunoblot analysis of proteic extracts from rainbow trout organs probed with anti-hBCL10. Lysates from HEK293T cells transfected with tBCL10 were used as a positive control (arrow).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372615&req=5

f0010: tBCL10 expression. (A–B) Immunoblot analysis of lysates from HEK293 cells transfected with the indicated expression vectors. Were indicated, prior to SDS–PAGE separation cell lysates were treated with 10 units of calf intestinal phosphatase (CIP) for 30 min at 37 °C. (C) Immunoblot analysis of proteic extracts from rainbow trout organs probed with anti-hBCL10. Lysates from HEK293T cells transfected with tBCL10 were used as a positive control (arrow).
Mentions: When analyzed in immunoblot assay, tBCL10 expressed in mammalian cells migrates as a 28 kDa protein (Fig. 2A). Interestingly, a rabbit antisera raised against hBCL10 also recognizes tBCL10 (Fig. 2A, right panel). In these expression experiments, we noticed that while hBCL10 migrates as a doublet on SDS–PAGE due to phosphorylation of the protein [25,26], tBCL10 occurs as a single band, suggesting that tBCL10 is not target of phosphorylation events. Indeed, experiments in which lysates were treated with phosphatase prior to immunoblot analysis confirm this possibility (Fig. 2B). Finally, an immunoblot assay carried out on proteic lysates extracted from various rainbow trout organs and tissues indicates that a band corresponding to the molecular weight of tBCL10 is expressed in spleen and, less intensely, in kidney (Fig. 2C). Additional bands with higher molecular weight are detectable, possibly corresponding to other BCL10 isoforms encoded by the genome of O.mykiss.

Bottom Line: Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20.Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein.This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

View Article: PubMed Central - PubMed

Affiliation: Biogem, Via Camporeale, Ariano Irpino (AV), Italy.

ABSTRACT
The complexes formed by BCL10, MALT1 and members of the family of CARMA proteins have recently been the focus of much attention because they represent a key mechanism for regulating activation of the transcription factor NF-κB. Here, we report the functional characterization of a novel isoform of BCL10 in the trout Oncorhynchus mykiss, which we named tBCL10. tBCL10 dimerizes, binds to components of the CBM complex and forms cytoplasmic filaments. Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20. Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein. This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

No MeSH data available.


Related in: MedlinePlus