In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants

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.


Anti-TE
and quantified in Figure 2F. For n=1, half volume of each sample was loaded following the loading plan shown on the upper corner of Figure S5. Only the conditions shown in green were taken into account for quantification. After transfer, each membrane was cut at the level of 75 kDa marker. The upper part of each membrane was blotted with an antimyc antibody and the lower part with an anti-GST antibody. The second half of each sample was loaded on two different gels following the same loading plan and transferred on membranes. Each membrane was then blotted with an anti-TE antibody.
The first membrane was stripped and blotted again with an anti-myc. For n=2 and n=3, each sample was loaded on 3 different gels and transferred on 3 different nitrocellulose membranes. Membranes were blotted with anti-myc, anti-GST and anti-TE antibodies, respectively.

Figure S4
Raw images corresponding to immunoblot data of Figure 2E and 2F

Figure S5
Raw images corresponding to immunoblot data of Figure  Raw images corresponding to immunoblot data of Figure 3D and 3E of the manuscript

Figure S7
Raw images corresponding to immunoblot data of Figure 3F, 3G and 3H of the manuscript Raw images corresponding to immunoblot data of Figure 4A and 4B of the manuscript  Raw images corresponding to immunoblot data of Figure 4C, 4D and 4E of the manuscript

Anti-GST (TIFA)
Anti-Myc   Figure 4F and quantified in Figure 4G. For each replicate, all samples were loaded on a gel and transferred on a nitrocellulose membrane. The membrane was then blotted with an anti-TE antibody.
The membrane was then stripped and cut as indicated by the scissor. The upper part of the membrane was blotted with an anti-myc antibody and the lower part with an anti-GST antibody.
Raw images corresponding to immunoblot data of Figure 4F and 4G of the manuscript n1 n3 (shown in manuscript) n2

Anti-myc (ALPK1)
Anti-TE Figure S12 kDa kDa kDa Figure S13: Images of the membranes corresponding to the immunoblots shown in Figure S1. For each replicate, half volume of each sample was loaded on a gel and transferred on a nitrocellulose membrane. The membrane was cut at the level of 75 kDa marker. The upper part of the membrane was blotted with an anti-myc antibody and the lower part with an anti-GST antibody. The second half of each sample was loaded on a separate gel and transferred on a membrane. The membrane was then blotted with an anti-pT9 antibody. The replicate shown in red was used for Figure 1D and the three replicates were used for quantification (red and blue rectangles).
Membrane images corresponding to the immunoblots shown in Figure S1 Anti-myc (ALPK1)

Anti-TIFA pT9
Anti-GST (TIFA) n1 n3 n2 (shown in manuscript) Figure S13  Figure S2. For each replicate, half volume of each sample was loaded on a gel and transferred on a nitrocellulose membrane. The membrane was cut at the level of 75 kDa marker. The upper part of the membrane was blotted with an anti-myc antibody and the lower part with an anti-GST antibody. The second half of each sample was loaded on a separate gel and transferred on a membrane. The membrane was then blotted with an anti-TE antibody.

Figure S14
Membrane images corresponding to the immunoblots shown in Figure S2

Figure S15
Membrane images corresponding to the immunoblots shown in Figure S3 Figure S4. For n=1, half volume of each sample was loaded following the loading plan shown on the upper corner of Figure S5. Only the conditions shown in green were taken into account for quantification. After transfer, each membrane was cut at the level of 75 kDa marker. The upper part of each membrane was blotted with an antimyc antibody and the lower part with an anti-GST antibody. The second half of each sample was loaded on two different gels following the same loading plan and transferred on membranes. Each membrane was then blotted with an anti-TE antibody.
The first membrane was stripped and blotted again with an anti-myc. For n=2 and n=3, each sample was loaded on 3 different gels and transferred on 3 different nitrocellulose membranes. Membranes were blotted with anti-myc, anti-GST and anti-TE antibodies, respectively.

Figure S16
Membrane images corresponding to the immunoblots shown in Figure S4 Anti-GST (TIFA) Anti-Myc (ALPK1) Anti-TE n1 n2 n3 (shown in manuscript) Figure S17: Images of the membranes corresponding to the immunoblots shown in Figure S5. For each replicate, half volume of each sample was loaded on a gel and transferred on a nitrocellulose membrane. The membrane was cut at the level of 75 kDa marker. The upper part of the membrane was blotted with an anti-myc antibody and the lower part with an anti-GST antibody. The second half of each sample was loaded on a separate gel and transferred on a membrane. The membrane was then blotted with an anti-TE antibody.

Figure S17
Membrane images corresponding to the immunoblots shown in Figure S5 Anti-GST (TIFA)

Anti-Myc (ALPK1)
Anti-TE n1 n3 n2 (shown in manuscript) Figure S18: Images of the membranes corresponding to the immunoblots shown in Figure S6. For n=1, half volume of each sample was loaded on a gel and transferred on a nitrocellulose membrane. The membrane was cut at the level of 75 kDa marker.
The upper part of the membrane was blotted with an anti-myc antibody and the lower part with an anti-GST antibody. The second half of each sample was loaded on a separate gel and transferred on a membrane. The membrane was then blotted with an anti-TE antibody. For n=2 and n=3, all samples were loaded on a gel and transferred on a nitrocellulose membrane. The membrane was then blotted with an anti-TE antibody. The membrane was then stripped and cut as indicated by the scissor. The