Physical and functional interactions between Drosophila TRAF2 and Pelle kinase contribute to Dorsal activation

Abstract

Signaling through the Toll receptor is required for dorsal/ventral polarity in Drosophila embryos, and also plays an evolutionarily conserved role in the immune response. Upon ligand binding, Toll appears to multimerize and activate the associated kinase, Pelle. However, the immediate downstream targets of Pelle have not been identified. Here we show that Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2), a homologue of human TRAF6, physically and functionally interacts with Pelle, and is phosphorylated by Pelle in vitro. Importantly, dTRAF2 and Pelle cooperate to activate Dorsal synergistically in cotransfected Schneider cells. Deletion of the C-terminal TRAF domain of dTRAF2 enhances Dorsal activation, perhaps reflecting the much stronger interaction of the mutant protein with phosphorylated, active Pelle. Taken together, our results indicate that Pelle and dTRAF2 physically and functionally interact, and that the TRAF domain acts as a regulator of this interaction. dTRAF2 thus appears to be a downstream target of Pelle. We discuss these results in the context of Toll signaling in flies and mammals.

The Drosophila Atypical Protein Kinase C-Ref(2)P Complex Constitutes a Conserved Module for Signaling in the Toll Pathway

“…10A demonstrate that the expression of DTRAF2, like that of Ref (2)P (Fig. 6), is sufficient to activate the Drosomycin promoter, in agreement with previously reported results (32). More importantly, the ability of increasing concentrations of Ref (2)P to activate the Drosomycin promoter is synergistically enhanced by the expression of low levels of DTRAF2, which alone do not significantly activate this parameter (Fig.…”

“…The human PKC cDNA was subcloned into the pPAC vector. HA-DTRAF2 was a generous gift from J. L. Manley (32). For expression in mammalian 293 cells, Myc-Ref(2)P, HA-Ref(2)P, and Flag-TRAF2 were constructed by subcloning the coding sequences into the pCDNA3 vector (Invitrogen).…”

“…In mammals, IRAK binds TRAF6, which in turn is responsible for NF-B and Jun kinase (JNK) activation (4,23). The Drosophila homologue of TRAF6 may be DTRAF2, which has recently been implicated in the activation of the Dorsal pathway (32).…”

Phylogeny of the TRAF/MATH Domain

“…TRAF6 orthologs have been found so far in insects, such as Drosophila (where is known as DTRAF2) 56 , mosquitoes and bees, as well as in vertebrates.…”

“…Drosophila's, TRAF6 ortholog, DTRAF2 participates in the control of gene expression by regulating the activation of the NFκB pathways 56,62,69 . Indeed, DTRAF2 is necessary for the expression of the anti-microbial peptides diptericin, diptericin-like protein and drosomycin in response to fungal and gram-positive bacteria infection, thus supporting a role for DTRAF2 (TRAF6) in innate immunity 62 .…”