Site-specific monoubiquitination of IkappaB kinase IKKbeta regulates its phosphorylation and persistent activation.
Transcription factor NF-kappaB governs the expression of multiple genes involved in cell growth, immunity, and inflammation. Nuclear translocation of NF-kappaB is regulated from the cytoplasm by IkappaB kinase-beta (IKKbeta), which earmarks inhibitors of NF-kappaB for polyubiquination and proteasome-mediated degradation. Activation of IKKbeta is contingent upon signal-induced phosphorylation of its T loop at Ser-177/Ser-181. T loop phosphorylation also renders IKKbeta a substrate for monoubiquitination in cells exposed to chronic activating cues, such as the Tax oncoprotein or sustained signaling through proinflammatory cytokine receptors. Here we provide evidence that the T loop-proximal residue Lys-163 in IKKbeta serves as a major site for signal-induced monoubiquitination with significant regulatory potential. Conservative replacement of Lys-163 with Arg yielded a monoubiquitination-defective mutant of IKKbeta that retains kinase activity in Tax-expressing cells but is impaired for activation mediated by chronic signaling from the type 1 receptor for tumor necrosis factor-alpha. Phosphopeptide mapping experiments revealed that the Lys-163 --> Arg mutation also interferes with proper in vivo but not in vitro phosphorylation of cytokine-responsive serine residues located in the distal C-terminal region of IKKbeta. Taken together, these data indicate that chronic phosphorylation of IKKbeta at Ser-177/Ser-181 leads to monoubiquitin attachment at nearby Lys-163, which in turn modulates the phosphorylation status of IKKbeta at select C-terminal serines. This mechanism for post-translational cross-talk may play an important role in the control of IKKbeta signaling during chronic inflammation.
Carter
Robert S
RS
Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Pennington
Kevin N
KN
Arrate
Pia
P
Oltz
Eugene M
EM
Ballard
Dean W
DW
D021581:Active Transport, Cell Nucleus; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002460:Cell Line; D049109:Cell Proliferation; D016207:Cytokines / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005822:Genetic Vectors; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D015151:Immunoblotting; D007249:Inflammation; D008239:Lysine / Q000737:chemistry; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism
D016207:Cytokines; D010455:Peptides; D025801:Ubiquitin; D012694:Serine; D001120:Arginine; D051550:I-kappa B Kinase; D008239:Lysine