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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 dimerizes and binds to CBM proteins. (A) HEK293 cells were transiently cotransfected with FLAG-tagged or HA-tagged versions of tBCL10 and hBCL10. 24 h later, cell lysates were immunoprecipitated with anti-FLAG mAb. Immunocomplexes were separated by SDS–PAGE and transferred onto membranes subsequently probed with anti-HA antisera. The right panel shows controls for immunoprecipitation specificity. (B) Lysates from HEK293 cells transfected with tBCL10 were analyzed for coprecipitating MALT1; (C) CARMA2sh and (D) CARMA3. (E) Over-exposure of immunoblot experiments described in (A) shows proteolytic processing of tBCL10.
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f0015: tBCL10 dimerizes and binds to CBM proteins. (A) HEK293 cells were transiently cotransfected with FLAG-tagged or HA-tagged versions of tBCL10 and hBCL10. 24 h later, cell lysates were immunoprecipitated with anti-FLAG mAb. Immunocomplexes were separated by SDS–PAGE and transferred onto membranes subsequently probed with anti-HA antisera. The right panel shows controls for immunoprecipitation specificity. (B) Lysates from HEK293 cells transfected with tBCL10 were analyzed for coprecipitating MALT1; (C) CARMA2sh and (D) CARMA3. (E) Over-exposure of immunoblot experiments described in (A) shows proteolytic processing of tBCL10.

Mentions: In mammals, BCL10 plays a crucial role in the signal transduction pathway that leads to activation of the transcription factor NF-κB [6,7]. hBCL10-mediated activation of NF-κB requires oligomerization of hBCL10, assembly of the CBM complex and triggering of unconventional ubiquitination events [7,27], which eventually result in the recruitment of the IKK complex [28]. Indeed, transfection experiments indicate that tBCL10 is able to dimerize both with itself and with hBCL10 (Fig. 3A). Furthermore, tBCL10 associates with human MALT1 (Fig. 3B), with human CARMA2sh[16] (Fig. 3C) and human CARMA3 (Fig. 3D). Finally, as activation of the CBM complex includes induction of the proteolytic activity of MALT1 which processes hBCL10 after R228 [29], we monitored the possible processing of tBCL10 when expressed in HEK293 cells. As shown in Fig. 3E, overexposure of the immunoblot of tBCL10 expressed in the HEK293 clearly shows the occurrence of a proteolytic processing of tBCL10.


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 dimerizes and binds to CBM proteins. (A) HEK293 cells were transiently cotransfected with FLAG-tagged or HA-tagged versions of tBCL10 and hBCL10. 24 h later, cell lysates were immunoprecipitated with anti-FLAG mAb. Immunocomplexes were separated by SDS–PAGE and transferred onto membranes subsequently probed with anti-HA antisera. The right panel shows controls for immunoprecipitation specificity. (B) Lysates from HEK293 cells transfected with tBCL10 were analyzed for coprecipitating MALT1; (C) CARMA2sh and (D) CARMA3. (E) Over-exposure of immunoblot experiments described in (A) shows proteolytic processing of tBCL10.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0015: tBCL10 dimerizes and binds to CBM proteins. (A) HEK293 cells were transiently cotransfected with FLAG-tagged or HA-tagged versions of tBCL10 and hBCL10. 24 h later, cell lysates were immunoprecipitated with anti-FLAG mAb. Immunocomplexes were separated by SDS–PAGE and transferred onto membranes subsequently probed with anti-HA antisera. The right panel shows controls for immunoprecipitation specificity. (B) Lysates from HEK293 cells transfected with tBCL10 were analyzed for coprecipitating MALT1; (C) CARMA2sh and (D) CARMA3. (E) Over-exposure of immunoblot experiments described in (A) shows proteolytic processing of tBCL10.
Mentions: In mammals, BCL10 plays a crucial role in the signal transduction pathway that leads to activation of the transcription factor NF-κB [6,7]. hBCL10-mediated activation of NF-κB requires oligomerization of hBCL10, assembly of the CBM complex and triggering of unconventional ubiquitination events [7,27], which eventually result in the recruitment of the IKK complex [28]. Indeed, transfection experiments indicate that tBCL10 is able to dimerize both with itself and with hBCL10 (Fig. 3A). Furthermore, tBCL10 associates with human MALT1 (Fig. 3B), with human CARMA2sh[16] (Fig. 3C) and human CARMA3 (Fig. 3D). Finally, as activation of the CBM complex includes induction of the proteolytic activity of MALT1 which processes hBCL10 after R228 [29], we monitored the possible processing of tBCL10 when expressed in HEK293 cells. As shown in Fig. 3E, overexposure of the immunoblot of tBCL10 expressed in the HEK293 clearly shows the occurrence of a proteolytic processing of tBCL10.

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