 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 TIFASMS, activation of NFB 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 vitrokinase assay based on the use of ATPS and protein-thiophosphorylation. We confirm that ALPK1 phosphorylates TIFA T9 and show that T2, T12 and T19 are. This article was authored by Diego Garcia-Weber and Sophie Dainjard, Veronica Teixeira and others. We are article.tv, links in the description below.