Background. Cellular biological responses to specific stimulation are determined by a balance among signaling pathways. Protein interactions are likely to modulate these pathways. Vaccinia-related kinase-2 (VRK2) is a novel human kinase that can modulate different signaling pathways. Principal findings. We report that in vivo, the activity of JIP1-JNK complexes is downregulated by VRK2 in response to interleukin-1β. Also the reduction of endogenous VRK2 with shRNA increases the transcriptional response to IL-1β. The JIP1 scaffold protein assembles three consecutive members of a given MAPK pathway forming signaling complexes and their signal can be modulated by interactions with regulatory proteins that remain to be identified. Knocking-down JIP1 with siRNA resulted in elimination of the AP1 transcriptional response to IL-1β. VRK2, a member of novel Ser-Thr kinase family, is able to stably interact with JIP1, TAK1 and MKK7, but not JNK, and can be isolated forming oligomeric complexes with different proportions of TAK1, MKK7β1 and JNK. JIP1 assembles all these proteins in an oligomeric signalosome. VRK2 binding to the JIP1 signalosome prevents the association of JNK and results in a reduction in its phosphorylation and downregulation of AP1-dependent transcription. Conclusions/Significance. This work suggests that the intracellular level of VRK2 protein can modulate the flow through a signaling pathway and alter the response from a receptor that can be distributed by more than one pathway, and thus contribute to the cellular specificity of the response by forming alternative signaling complexes. Furthermore, the effect might be more general and affect other signaling routes assembled on the JIP1 scaffold protein for which a model is proposed.S.B., M. S-G, and C.R.S. have predoctoral fellowships from Ministerio de Educación y Ciencia, CSIC (Spain) and Fundação para a Ciência e a Tecnologia (Portugal) respectively. This work was funded by grants from Ministerio de Educación y Ciencia (SAF2004-02900, SAF2007-60242 and Consolider CSD-2007-0017), Fundación de Investigación Médica MM and Federación de Cajas de Ahorro de Castilla y León to P.A.L.Peer reviewed