Mixed-lineage kinase 3 delivers CD3/CD28-derived signals into the I kappa B kinase complex

The phosphorylation of I kappa B by the multiprotein I kappa B kinase compl ex (IKC) precedes the activation of transcription factor NF-kappa B, a key regulator of the inflammatory response. Here we identified the mixed-lineag e group kinase 3 (MLK3) as an activator of NF-kappa B. Expression of the wi ld-type form of this mitogen-activated protein kinase kinase kinase (MAPKKK ) induced nuclear immigration, DNA binding, and transcriptional activity of NF-kappa B. MLM3 directly phosphorylated and thus activated I kappa B kina se alpha (IKK alpha) and IKK beta, revealing its function as an I kappa B k inase kinase (IKKK). MLK3 cooperated with the other two IKKKs, MEKK1 and NF -kappa B-inducing kinase, in the induction of IKK activity. MLK3 bound to c omponents of the IKC in vivo. This protein-protein interaction was dependen t on the central leucine zipper region of MLK3. A kinase-deficient version of MLK3 strongly impaired NF-kappa B-dependent transcription induced by T-c ell costimulation but not in response to tumor necrosis factor alpha or int erleukin-l. Accordingly, endogenous MLK3 was phosphorylated and activated b y T-cell costimulation but not by treatment of cells with tumor necrosis fa ctor alpha or interleukin-1. A dominant negative version of MLM3 inhibited NF-kappa B- and CD28RE/AP-dependent transcription elicited by the Rho famil y GTPases Rac and Cdc42, thereby providing a novel link between these GTPas es and the IKC.