The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-κB via the IκB kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-κB activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-κB by the dsRNA-dependent protein kinase.This investigation was supported by research grants BMC2002- 03246 and BIO2000-0340-P4 from Spain and QLK2-CT2002-00954 from the European Union to M.E., by grants SAF2000-0028 and FIPSE to J.A., and by the Fundación Ramón Areces (P.G.-P.). H.N. is supported by a grant from the Human Frontier Science Program. M.A.G. and J.G. were supported by fellowships from the Ministerio de Ciencia y Tecnología, Madrid, Spain.Peer reviewed