Control of mitogen-activated protein kinase (MAPK) cascades is central to regulation of many cellular responses. We describe here human tribbles homologues (Htrbs) that control MAPK activity. MAPK kinases interact with Trbs and regulate their steady state levels. Further, Trbs selectively regulate the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 MAPK with different relative levels of activity for the three classes of MAPK observed depending on the level of Trb expression. These results suggest that Trbs control both the extent and the specificity of MAPK kinase activation of MAPK.

Type I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is reflected in similarity in signaling: all members tested couple to activation of NFκB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFκB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways.

The internalization and intracellular transport of IL-1 and its receptor were examined in the murine T cell line EL-4. For 4 h after internalization intracellular 125I-IL-1 alpha remains bound to its receptor without degradation. Electron microscope autoradiography demonstrates that internalized IL-1 accumulates in purified nuclei. The IL-1 extracted from these nuclei is still bound to receptor. As no receptors for IL-1 were detected in untreated nuclei, these results suggest IL-1 driven translocation of the cell surface IL-1R complex to the nucleus. IL-1R internalization was correlated with IL-1 signal transduction events required to induce growth factor production from several subclones of EL-4 cells. The subsequent transport of the internalized IL-1R complex to the nucleus suggests the possibility for a nuclear site for IL-1R signaling.

Activation of the nuclear factor κB (NFκB) transcription factor is intimately associated with its translocation from the cytoplasm to the nucleus. Using the nuclear export inhibitor leptomycin B, we demonstrate shuttling of the RELA subunit of NFκB and the inhibitory subunit IκBα between these two compartments in unstimulated cells. Determination of the kinetics of nuclear entry shows marked differences for the two components; the entry of IκBα occurs more rapidly than RELA. The shuttling is suggested to be a consequence of the cytoplasmic dissociation of the NFκB·IκB complex rather than its direct nuclear import or degradation and resynthesis of IκBα. Using previously published kinetic data, this proposition is born out by the deduction that 17% of NFκB is not complexed to IκBα in a resting cell. A numerical model is presented to validate the proposed regulation of NFκB subcellular localization consequent in part on the nuclear export function and in part on the cytoplasmic retention function of IκBα. We suggest that the non-saturated interaction of NFκB with the inhibitor may enhance the specificity of action of IκB proteins on different NFκB dimers and allow additional modes of regulation of IκB function.