N- and C-Terminal Sequences Control Degradation of MAD3/IkBa in Response to Inducers of NF-kB Activity

The proteolytic degradation of the inhibitory protein MAD3/IkBain response to extracellular stimulation is a prerequisite step in the activation of the transcription factor NF-kB. Analysis of the expression of human IkBaprotein in stable transfectants of mouse 70Z/3 cells shows that, as for the endogenous murine protein, exogenous IkBais degraded in response to inducers of NF-kB activity, such as phorbol myristate acetate or lipopolysaccharide. In addition, pretreatment of the cells with the proteasome inhibitor N-Ac-Leu-Leu-norleucinal inhibits this ligand-induced degradation and, in agreement with previous studies, stabilizes a hyperphosphorylatedformofthehumanIkBaprotein.Byexpressingmutantformsofthehumanproteininthiscell line,wehavebeenabletodelineatethesequencesresponsibleforboththeligand-inducedphosphorylationand the degradation of IkBa. Our results show that deletion of the C terminus of the IkBamolecule up to amino acid 279 abolishes constitutive but not ligand-inducible phosphorylation and inhibits ligand-inducible degradation. Further analysis reveals that the inducible phosphorylation of IkBa maps to two serines in the N terminus of the protein (residues 32 and 36) and that the mutation of either residue is sufficient to abolish ligand-induced degradation, whereas both residues must be mutated to abolish inducible phosphorylation of the protein. We propose that treatment of 70Z/3 cells with either phorbol myristate acetate or lipopolysaccharide induces a kinase activity which phosphorylates serines 32 and 36 and that these phosphorylations target the protein for rapid proteolytic degradation, possibly by the ubiquitin-26S proteasome pathway, thus allowing NF-kB to translocate to the nucleus and to activate gene expression. The Rel/NF-kB family of proteins (p50, p52, p65/RelA, cRel, and RelB) bind specifically as homo- or heterodimers to kB motifs located in the promoters and enhancers of a large number of genes that control various aspects of the immune andinflammatoryresponses(includingthemajorhistocompatibility complex class I genes, the immunoglobulin klight chain, interleukin 2 and its receptor, interleukins 6 and 8, granulocyte-macrophage colony-stimulating factor, beta interferon, and T-cell receptor bchain) as well as those of several viruses, including human immunodeficiency virus types 1 and 2 and cytomegalovirus (for recent reviews, see references 2 and 32). In most cell types, NF-kB is maintained in an inactive, cytoplasmic state in complexes with members of the IkB family, which includes IkBa ,I k B b, and IkBg (only B cells and some cells of the monocyte or macrophage lineage exhibit constitutive nuclear NF-kB activity). These inhibitors all contain multiple copies of a motif, the ankyrin repeat, which interact with and inhibit the nuclear localization and DNA binding of the Rel/NF-kB proteins (for a review, see reference 3). NF-k Bi s induced by a wide variety of stimuli (including phorbol myristate acetate [PMA], tumor necrosis factor, interleukin 1, lipopolysaccharide [LPS], viral infection, and many mitogens and cytokines) through a mechanism involving inactivation of