ALPK1 mutants causing ROSAH syndrome or Spiradenoma are activated by human nucleotide sugars

Abstract

The atypical protein kinase ALPK1 is activated by the bacterial nucleotide sugar ADP-heptose and phosphorylates TIFA to switch on a signaling pathway that combats microbial infection. In contrast, ALPK1 mutations cause two human diseases: the ALPK1[T237M] and ALPK1[Y254C] mutations underlie ROSAH syndrome (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis, and migraine headache), while the ALPK1[V1092A] mutation accounts for 45% of spiradenoma and 30% of spiradenocarcinoma cases studied. In this study, we demonstrate that unlike wild-type (WT) ALPK1, the disease-causing ALPK1 mutants trigger the TIFA-dependent activation of an NF-κB/activator protein 1 reporter gene in the absence of ADP-heptose, which can be suppressed by either of two additional mutations in the ADP-heptose binding site that prevent the activation of WT ALPK1 by ADP-heptose. These observations are explained by our key finding that although ALPK1[T237M] and ALPK1[V1092A] are activated by bacterial ADP-heptose, they can also be activated by nucleotide sugars present in human cells (UDP-mannose, ADP-ribose, and cyclic ADP-ribose) which can be prevented by disruption of the ADP-heptose binding site. The ALPK1[V1092A] mutant was also activated by GDP-mannose, which did not activate ALPK1[T237M]. These are new examples of disease-causing mutations permitting the allosteric activation of an enzyme by endogenous molecules that the WT enzyme does not respond to. We propose that the loss of the specificity of ALPK1 for bacterial ADP-heptose underlies ROSAH syndrome and spiradenoma/spiradenocarcinoma caused by ALPK1 mutation.

Keywords: ALPK1; ROSAH; TIFA; nucleotide sugar; spiradenoma.

Fig. 1.

Outline of the ADP-heptose-ALPK1-TIFA signalling pathway. The bacterial nucleotide sugar ADP-heptose crosses the plasma membrane of human cells, where it binds to and activates the cytosolic human protein kinase ALPK1, allowing it to phosphorylate TIFA at Thr9. This induces the polymerisation of TIFA and the recruitment of the E3 ligases c-IAP1 (recruited by TRAF2) and TRAF6, which produce the Lys63-linked ubiquitin chains (K63-ubiquitin) required to activate the TAK1 kinase complex (3). TAK1 then activates JNK and the IKKβ component of the canonical IKK complex. IKKβ activation also requires the formation of Met1-linked ubiquitin chains (M1-ubiquitin) produced by the E3 ligase HOIP, a component of the LUBAC. How HOIP is recruited to the signalling complex is not fully understood, but the Npl4 zinc finger domain of HOIP interacts selectively with Lys63-linked ubiquitin oligomers (9). Once activated, JNK and IKKβ activate the transcription factors AP-1 and NF-κB, respectively, switching on the transcription of genes encoding inflammatory mediators. E3 ligases are highlighted in blue, protein kinases in pink, and noncatalytic components in grey.

Fig. 2.

ADP-heptose-dependent and -independent NF-κB/AP-1 gene transcription in cells expressing WT ALPK1 and disease-causing mutants of ALPK1. (A and B) WT (A) or ALPK1 KO HEK293-Blue cells (B) were stimulated in the absence (black line) or presence (blue line) of ADP-heptose (ADP-H) and the activation of NF-κB/AP-1-dependent gene transcription measured after the times indicated (Methods). (C) WT cells were incubated for 1 h with 5 µM NG25 (TAK1 inhibitor, denoted TAK1 i), 10 µM BI605906 (IKKβ inhibitor, denoted IKKβ i), 5 µM JNK-IN-8 (JNK inhibitor, denoted JNK i) or a combination of 10 µM BI605906 and 5 µM JNK-IN-8 (IKKβ i + JNK i), as indicated. The cells were then incubated with (blue bars) or without (grey bars) ADP-H and the activation of gene transcription measured after 24 h. (D–G) ALPK1 KO cells were transfected with a plasmid encoding WT ALPK1 (D), ALPK1[T237M] (E), ALPK1[Y254C] (F) or ALPK1[V1092A] (G) as indicated and 24 h later were analysed as in B. (H) Cell extracts (15 µg of protein) from ALPK1 KO cells transfected with empty vector (lane 1), FLAG-tagged WT ALPK1 (lane 2), or the indicated FLAG-tagged ALPK1 mutant (lanes 3–5) were subjected to SDS-PAGE and immunoblotted using the antibodies indicated. (A–G) The results are expressed as mean ± SD from an experiment using three or four dishes of independently transfected cells and the experiment was repeated twice with similar results each time.

Fig. 3.

The cellular activity of ALPK1 disease mutants in the absence of ADP-heptose is dependent on the kinase activity of ALPK1, the expression of TIFA and an intact ADP-heptose binding site. (A) ALPK1 KO cells were transfected with plasmids encoding WT ALPK1 or the indicated ALPK1 mutants and 24 h later incubated with (blue bars) or without (gray bars) ADP-H and the activation of gene transcription measured after a further 24 h (Methods). (B) ALPK1 KO or two different clones of ALPK1/TIFA DKO cells (clones A and B) were transfected with the different plasmids indicated and analysed as in A. (C) As in A, but transfection was performed with the different plasmids indicated. (A–C) The results are expressed as mean ± SD from an experiment using three or five dishes of independently transfected cells and the experiment was repeated twice with similar results each time.

Fig. 4.

ALPK1 disease mutants, but not the WT protein, are activated by human nucleotide sugars in vitro. (A and B) FLAG-tagged WT ALPK1 (WT, gray bars), ALPK1[T237M] (T237M, blue bars) or ALPK1[V1092A] (V1092A, pink bars) were immunoprecipitated from cell extracts and assayed for 20 min in the absence or presence of ADP-heptose (1 µM) or the nucleotide sugars indicated (100 µM) and the incorporation of ³²P-radioactivity into GST-TIFA quantified (Methods). (C) As in A and B, except that the activity of ALPK1[V1092A] was compared to ALPK1[V1092A/R153A] (V1092A/R153A, unfilled bars). (D) As in A and B, except that the activity of ALPK1[T237M] was compared to ALPK1[T237M/R153A] (T237M/R153A, unfilled bars). (A–D) The nucleotide sugars are abbrievated as follows: ADP-heptose (ADP-H), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), UDP-mannose (UDP-Man), ADP-ribose (ADP-R), cyclic ADP-ribose (cADP-R), ADP-glucose (ADP-Glu), UDP-glucose (UDP-Glu), UDP-glucuronate (UDP-GluA), UDP-N-acetyl-glucosamine (UDP-GlcNAc), UDP-N-acetyl-galactosamine (UDP-GalNAc), and UDP-xylose (UDP-Xyl). The results are expressed as mean ± SEM from a total of 4 kinase assays (two experiments, each performed in duplicate).