Bruton's tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids

The Ras-GTPase activating protein SH3 domain-binding protein 1 (G3BP1) plays a critical role in the formation of classical and antiviral stress granules in stressed and virus-infected eukaryotic cells, respectively. While G3BP1 is known to be phosphorylated at serine residues which could affect stre...

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Main Authors: Kim, Susana S-Y., Sim, Don C. N., Carissimo, Guillaume, Lim, Hong-Hwa, Lam, Kong-Peng
Other Authors: School of Biological Sciences
Format: Article
Language:English
Published: 2022
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Online Access:https://hdl.handle.net/10356/163921
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spelling sg-ntu-dr.10356-1639212023-02-28T17:13:16Z Bruton's tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids Kim, Susana S-Y. Sim, Don C. N. Carissimo, Guillaume Lim, Hong-Hwa Lam, Kong-Peng School of Biological Sciences Singapore Immunology Network, A*STAR National University of Singapore Science::Biological sciences Endoribonuclease G3BP Rasputin The Ras-GTPase activating protein SH3 domain-binding protein 1 (G3BP1) plays a critical role in the formation of classical and antiviral stress granules in stressed and virus-infected eukaryotic cells, respectively. While G3BP1 is known to be phosphorylated at serine residues which could affect stress granule assembly, whether G3BP1 is phosphorylated at tyrosine residues and how this posttranslational modification might affect its functions is less clear. Here, we show using immunoprecipitation and immunoblotting studies with 4G10 antibody that G3BP1 is tyrosine-phosphorylated when cells are stimulated with the synthetic double-stranded RNA analog polyinosinic:polycytidylic acid to mimic viral infection. We further demonstrate via co-immunoprecipitation and inhibitor studies that Bruton's tyrosine kinase (BTK) binds and phosphorylates G3BP1. The nuclear transport factor 2-like domain of G3BP1 was previously shown to be critical for its self-association to form stress granules. Our mass spectrometry, mutational and biochemical cross-linking analyses indicate that the tyrosine-40 residue in this domain is phosphorylated by BTK and critical for G3BP1 oligomerization. Furthermore, as visualized via confocal microscopy, pretreatment of cells with the BTK inhibitor LFM-A13 or genetic deletion of the btk gene or mutation of G3BP1-Y40 residue to alanine or phenylalanine all significantly attenuated the formation of antiviral stress granule aggregates upon polyinosinic:polycytidylic acid treatment. Taken together, our data indicate that BTK phosphorylation of G3BP1 induces G3BP1 oligomerization and facilitates the condensation of ribonucleoprotein complexes into macromolecular aggregates. Agency for Science, Technology and Research (A*STAR) National Medical Research Council (NMRC) Published version This project was funded by National Medical Research Council (NMRC) OF-YIRG19MAY0014 (S. S.-Y. K.) and the Agency for Science, Technology, and Research (A*STAR) (L. K.-P.). 2022-12-22T02:09:32Z 2022-12-22T02:09:32Z 2022 Journal Article Kim, S. S., Sim, D. C. N., Carissimo, G., Lim, H. & Lam, K. (2022). Bruton's tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids. Journal of Biological Chemistry, 298(8), 102231-. https://dx.doi.org/10.1016/j.jbc.2022.102231 0021-9258 https://hdl.handle.net/10356/163921 10.1016/j.jbc.2022.102231 35798143 2-s2.0-85135520352 8 298 102231 en OF-YIRG19MAY-0014 Journal of Biological Chemistry © 2022 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
Endoribonuclease G3BP
Rasputin
spellingShingle Science::Biological sciences
Endoribonuclease G3BP
Rasputin
Kim, Susana S-Y.
Sim, Don C. N.
Carissimo, Guillaume
Lim, Hong-Hwa
Lam, Kong-Peng
Bruton's tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids
description The Ras-GTPase activating protein SH3 domain-binding protein 1 (G3BP1) plays a critical role in the formation of classical and antiviral stress granules in stressed and virus-infected eukaryotic cells, respectively. While G3BP1 is known to be phosphorylated at serine residues which could affect stress granule assembly, whether G3BP1 is phosphorylated at tyrosine residues and how this posttranslational modification might affect its functions is less clear. Here, we show using immunoprecipitation and immunoblotting studies with 4G10 antibody that G3BP1 is tyrosine-phosphorylated when cells are stimulated with the synthetic double-stranded RNA analog polyinosinic:polycytidylic acid to mimic viral infection. We further demonstrate via co-immunoprecipitation and inhibitor studies that Bruton's tyrosine kinase (BTK) binds and phosphorylates G3BP1. The nuclear transport factor 2-like domain of G3BP1 was previously shown to be critical for its self-association to form stress granules. Our mass spectrometry, mutational and biochemical cross-linking analyses indicate that the tyrosine-40 residue in this domain is phosphorylated by BTK and critical for G3BP1 oligomerization. Furthermore, as visualized via confocal microscopy, pretreatment of cells with the BTK inhibitor LFM-A13 or genetic deletion of the btk gene or mutation of G3BP1-Y40 residue to alanine or phenylalanine all significantly attenuated the formation of antiviral stress granule aggregates upon polyinosinic:polycytidylic acid treatment. Taken together, our data indicate that BTK phosphorylation of G3BP1 induces G3BP1 oligomerization and facilitates the condensation of ribonucleoprotein complexes into macromolecular aggregates.
author2 School of Biological Sciences
author_facet School of Biological Sciences
Kim, Susana S-Y.
Sim, Don C. N.
Carissimo, Guillaume
Lim, Hong-Hwa
Lam, Kong-Peng
format Article
author Kim, Susana S-Y.
Sim, Don C. N.
Carissimo, Guillaume
Lim, Hong-Hwa
Lam, Kong-Peng
author_sort Kim, Susana S-Y.
title Bruton's tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids
title_short Bruton's tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids
title_full Bruton's tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids
title_fullStr Bruton's tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids
title_full_unstemmed Bruton's tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids
title_sort bruton's tyrosine kinase phosphorylates scaffolding and rna-binding protein g3bp1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids
publishDate 2022
url https://hdl.handle.net/10356/163921
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