Abstract
The complexes formed by BCL10, MALT1 and specific members of the family of CARMA proteins (CBM complex), have recently focused much attention because they represent a central hub regulating activation of the transcription factor NF-κB following various cellular stimulations. In this manuscript, we report the functional characterization of a Danio rerio 241 amino acids polypeptide ortholog of the Caspase recruiting domain (CARD)-containing protein BCL10. Biochemical studies show that zebrafish Bcl10 (zBcl10) dimerizes and binds to components of the CBM complex. Fluorescence microscopy observations demonstrate that zBcl10 forms cytoplasmic filaments similar to that formed by human BCL10 (hBCL10). Functionally, in human cells zBcl10 is more effective in activating NF-κB compared to hBCL10, possibly due to the lack of carboxy-terminal inhibitory serine residues present in the human protein. Also, depletion experiments carried out through expression of short hairpin RNAs targeting hBCL10 indicate that zBcl10 can functionally replace the human protein. Finally, we show that the zebrafish cell line PAC2 is suitable to carry out reporter assays for monitoring the activation state of NF- kB transcription factor. In conclusion, this work shows that zebrafish may excellently serve as a model organism to study complex and intricate signal transduction pathways, such as those that control NF-κB activation.
Highlights
NF-κB is an inducible and ubiquitously expressed transcription factor for genes involved in immune and inflammatory responses, cell survival, cell adhesion, differentiation, and growth [1, 2]
A paradigmatic example of this is given by the human CARDcontaining protein BCL10, a 233 amino acids protein initially identified by functional cloning approach from mucosa-associated lymphoid tissue (MALT) lymphoma cells [3, 4]
The major amino acidic differences between the two proteins are located at the carboxy-terminal of the polypeptides, whereas the amino-terminal Caspase recruiting domain (CARD) domains of zebrafish Bcl10 (zBcl10) and human BCL10 (hBCL10) share 62% identity (Table 1)
Summary
NF-κB is an inducible and ubiquitously expressed transcription factor for genes involved in immune and inflammatory responses, cell survival, cell adhesion, differentiation, and growth [1, 2]. Given that NF-κB transcribes genes that generally control both innate and acquired immune response and genes that play a positive effect on cell survival and proliferation, disregulation of the mechanisms controlling its activation often results in immunoproliferative and inflammatory phenotypes [1, 2]. Functional Characterization of Zebrafish Bcl of a translocation, in a subset of MALT B cell lymphomas BCL10 was overexpressed, resulting in an altered, constitutive activation of NF-κB that was eventually responsible for the neoplastic transformation [3, 4]. BCL10 was independently cloned in other laboratories for its ability to activate the transcription factor NF-κB [5,6,7,8,9,10]
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