Raf Kinase Inhibitor Protein Interacts with NF-κB-Inducing Kinase and TAK1 and Inhibits NF-κB Activation

ABSTRACT The Raf kinase inhibitor protein (RKIP) acts as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by Raf-1. RKIP inhibits the phosphorylation of MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by disrupting the interaction between these two kinases. We show here that RKIP also antagonizes the signal transduction pathways that mediate the activation of the transcription factor nuclear factor kappa B (NF-κB) in response to stimulation with tumor necrosis factor alpha (TNF-α) or interleukin 1 beta. Modulation of RKIP expression levels affected NF-κB signaling independent of the MAPK pathway. Genetic epistasis analysis involving the ectopic expression of kinases acting in the NF-κB pathway indicated that RKIP acts upstream of the kinase complex that mediates the phosphorylation and inactivation of the inhibitor of NF-κB (IκB). In vitro kinase assays showed that RKIP antagonizes the activation of the IκB kinase (IKK) activity elicited by TNF-α. RKIP physically interacted with four kinases of the NF-κB activation pathway, NF-κB-inducing kinase, transforming growth factor beta-activated kinase 1, IKKα, and IKKβ. This mode of action bears striking similarities to the interactions of RKIP with Raf-1 and MEK1 in the MAPK pathway. Emerging data from diverse organisms suggest that RKIP and RKIP-related proteins represent a new and evolutionarily highly conserved family of protein kinase regulators. Since the MAPK and NF-κB pathways have physiologically distinct roles, the function of RKIP may be, in part, to coordinate the regulation of these pathways.