In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants
Abstract Alpha-protein kinase 1 (ALPK1) is a pathogen recognition receptor that detects ADP-heptose (ADPH), a lipopolysaccharide biosynthesis intermediate, recently described as a pathogen-associated molecular pattern in Gram-negative bacteria. ADPH binding to ALPK1 activates its kinase domain and t...
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| Format: | Article |
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Nature Portfolio
2023-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-33459-7 |
| _version_ | 1797840997339627520 |
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| author | Diego García-Weber Anne-Sophie Dangeard Veronica Teixeira Martina Hauke Alexis Carreaux Christine Josenhans Cécile Arrieumerlou |
| author_facet | Diego García-Weber Anne-Sophie Dangeard Veronica Teixeira Martina Hauke Alexis Carreaux Christine Josenhans Cécile Arrieumerlou |
| author_sort | Diego García-Weber |
| collection | DOAJ |
| description | Abstract Alpha-protein kinase 1 (ALPK1) is a pathogen recognition receptor that detects ADP-heptose (ADPH), a lipopolysaccharide biosynthesis intermediate, recently described as a pathogen-associated molecular pattern in Gram-negative bacteria. ADPH binding to ALPK1 activates its kinase domain and triggers TIFA phosphorylation on threonine 9. This leads to the assembly of large TIFA oligomers called TIFAsomes, activation of NF-κB and pro-inflammatory gene expression. Furthermore, mutations in ALPK1 are associated with inflammatory syndromes and cancers. While this kinase is of increasing medical interest, its activity in infectious or non-infectious diseases remains poorly characterized. Here, we use a non-radioactive ALPK1 in vitro kinase assay based on the use of ATPγS and protein thiophosphorylation. We confirm that ALPK1 phosphorylates TIFA T9 and show that T2, T12 and T19 are also weakly phosphorylated by ALPK1. Interestingly, we find that ALPK1 itself is phosphorylated in response to ADPH recognition during Shigella flexneri and Helicobacter pylori infection and that disease-associated ALPK1 mutants exhibit altered kinase activity. In particular, T237M and V1092A mutations associated with ROSAH syndrome and spiradenoma/spiradenocarcinoma respectively, exhibit enhanced ADPH-induced kinase activity and constitutive assembly of TIFAsomes. Altogether, this study provides new insights into the ADPH sensing pathway and disease-associated ALPK1 mutants. |
| first_indexed | 2024-04-09T16:23:46Z |
| format | Article |
| id | doaj.art-434deacb5e94426db8c6d6142e4b9424 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-09T16:23:46Z |
| publishDate | 2023-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-434deacb5e94426db8c6d6142e4b94242023-04-23T11:17:24ZengNature PortfolioScientific Reports2045-23222023-04-0113111410.1038/s41598-023-33459-7In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutantsDiego García-Weber0Anne-Sophie Dangeard1Veronica Teixeira2Martina Hauke3Alexis Carreaux4Christine Josenhans5Cécile Arrieumerlou6Université Paris Cité, CNRS, INSERM, Institut CochinUniversité Paris Cité, CNRS, INSERM, Institut CochinUniversité Paris Cité, CNRS, INSERM, Institut CochinMax von Pettenkofer Institute, Ludwig Maximilians Universität MünchenUniversité Paris Cité, CNRS, INSERM, Institut CochinMax von Pettenkofer Institute, Ludwig Maximilians Universität MünchenUniversité Paris Cité, CNRS, INSERM, Institut CochinAbstract Alpha-protein kinase 1 (ALPK1) is a pathogen recognition receptor that detects ADP-heptose (ADPH), a lipopolysaccharide biosynthesis intermediate, recently described as a pathogen-associated molecular pattern in Gram-negative bacteria. ADPH binding to ALPK1 activates its kinase domain and triggers TIFA phosphorylation on threonine 9. This leads to the assembly of large TIFA oligomers called TIFAsomes, activation of NF-κB and pro-inflammatory gene expression. Furthermore, mutations in ALPK1 are associated with inflammatory syndromes and cancers. While this kinase is of increasing medical interest, its activity in infectious or non-infectious diseases remains poorly characterized. Here, we use a non-radioactive ALPK1 in vitro kinase assay based on the use of ATPγS and protein thiophosphorylation. We confirm that ALPK1 phosphorylates TIFA T9 and show that T2, T12 and T19 are also weakly phosphorylated by ALPK1. Interestingly, we find that ALPK1 itself is phosphorylated in response to ADPH recognition during Shigella flexneri and Helicobacter pylori infection and that disease-associated ALPK1 mutants exhibit altered kinase activity. In particular, T237M and V1092A mutations associated with ROSAH syndrome and spiradenoma/spiradenocarcinoma respectively, exhibit enhanced ADPH-induced kinase activity and constitutive assembly of TIFAsomes. Altogether, this study provides new insights into the ADPH sensing pathway and disease-associated ALPK1 mutants.https://doi.org/10.1038/s41598-023-33459-7 |
| spellingShingle | Diego García-Weber Anne-Sophie Dangeard Veronica Teixeira Martina Hauke Alexis Carreaux Christine Josenhans Cécile Arrieumerlou In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants Scientific Reports |
| title | In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants |
| title_full | In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants |
| title_fullStr | In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants |
| title_full_unstemmed | In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants |
| title_short | In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants |
| title_sort | in vitro kinase assay reveals adp heptose dependent alpk1 autophosphorylation and altered kinase activity of disease associated alpk1 mutants |
| url | https://doi.org/10.1038/s41598-023-33459-7 |
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