Tube Is an IRAK-4 homolog in a Toll pathway adapted for development and immunity

Abstract

Acting through the Pelle and IRAK family of protein kinases, Toll receptors mediate innate immune responses in animals ranging from insects to humans. In flies, the Toll pathway also functions in patterning of the syncytial embryo and requires Tube, a Drosophila -specific adaptor protein lacking a catalytic domain. Here we provide evidence that the Tube, Pelle, and IRAK proteins originated from a common ancestral gene. Following gene duplication, IRAK-4, Tube-like kinases, and Tube diverged from IRAK-1, Pelle, and related kinases. Remarkably, the function of Tube and Pelle in Drosophila embryos can be reconstituted in a chimera modeled on the predicted progenitor gene. In addition, a divergent property of downstream transcription factors was correlated with developmental function. Together, these studies reveal previously unrecognized parallels in Toll signaling in fly and human innate immunity and shed light on the evolution of pathway organization and function.

Keywords: Death domain; Drosophila; Evolution; Gene duplication; NF-κB; Signal transduction.

Fig. 1

TTLK proteins, Pelle and IRAK are members of a kinase superfamily. a Diagram of the carbon backbone of the D. melanogaster Tube death domain; side chains required for binding to MyD88 (blue) and Pelle (red) are represented in space-filling form. b Location within the bilaterian animal phylogenetic tree of species encoding identified Tube orthologs. c Clustal W alignment of the death domains of TTLK and Pelle proteins; numbers indicate position in the polypeptide sequence. The TTLK death domains exhibit complete conservation of residues required for binding to MyD88 (blue), as well as strong conservation of residues required for binding to Pelle (red).

Fig. 2

Scale diagram of the structural elements comprising TTLK and Pelle proteins. The asterisk indicates that the A. mellifera Pelle sequence is incomplete.

Fig. 3

Tube-like kinases, but not Pelle proteins, are RD kinases. Alignment of sequence subdomains VI and VII from TTLK and Pelle proteins. Arrow indicates position of RD dipeptide that is hallmark of kinases regulated by activation loop phosphorylation.

Fig. 4

a Tube-Pelle chimera mediates robust, spatially regulated Toll signaling, a Diagram of the Tube-Pll^C and Tube^N-Pll^C chimeras. The Tube-Pll^C construct encodes full-length Tube (amino acids 1–462) fused to amino acids 130–501 of Pelle. The Tube^N-Pll^C construct encodes amino acids 1–185 of Tube fused to the same Pelle fragment. b–d Darkfield micrographs of cuticle preparations from an uninjected embryo (b) and embryos injected with 1 mg/ml RNA from an in vitro transcription reaction (c, d). VM = Intact or fragmented vitelline membrane that encases the developing embryo. b Uninjected tub^null pll^null embryo. c tub^null pll^nul1 embryo injected with Tube-Pll^C RNA. White arrowheads point to the properly spaced bands of ventral denticles; a black arrowhead indicates the filzkörper apparatus, d tub^null pll^null embryo injected with Tube^N-Pll^C RNA. No Toll-dependent patterning elements are observed.

Fig. 5

Tube interacts with dorsal, but not the related NF-κB transcription factors Dif and Relish. S2 cells were transfected with epitope-tagged Tube – either Tube-myc or Tube^C-myc (amino acids 236–462 of Tube) – in combination with plasmids expressing V5-tagged forms of the proteins listed at the top of the figure. Immunoprecipitates were prepared with α-Tube antiserum. Tube proteins were detected with α-myc antibody (c); co-precipitating proteins were detected with α-V5 antibody (b). Samples of each cell lysate were probed with α-V5 antibody to assay expression of each V5-tagged protein (a). Dif was expressed under the control of two alternative 5′UTR sequences, which result in different levels of protein (top panel; lanes 4, 5 and 9, 10). The Rel^N construct (lanes 7 and 12) encodes amino acids 1–478 of Relish.

Fig. 6

Conserved Toll-proximal signaling mechanism. Model for organization of signaling complexes immediately downstream of Toll receptors in flies and humans.