In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B.
NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.
Alkalay
I
I
Lautenberg Center for General and Tumor Immunology, Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Yaron
A
A
Hatzubai
A
A
Jung
S
S
Avraham
A
A
Gerlitz
O
O
Pashut-Lavon
I
I
Ben-Neriah
Y
Y
D000595:Amino Acid Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020672:I-kappa B Proteins; D015151:Immunoblotting; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D051996:Transcription Factor RelA; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007976:Leupeptins; D046911:Macromolecular Substances; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009842:Oligopeptides; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; C032854:leupeptin