INVOLVEMENT OF VALOSIN-CONTAINING PROTEIN, AN ATPASE COPURIFIED WITH I-KAPPA-B-ALPHA AND 26-S PROTEASOME, IN UBIQUITIN-PROTEASOME-MEDIATED DEGRADATION OF I-KAPPA-B-ALPHA

The inactivation of the prototype NF-kappa B inhibitor, I kappa B alph a, occurs through a series of ordered processes including phosphorylat ion, ubiquitin conjugation, and proteasome-mediated degradation. We id entify valosin-containing protein (VCP), an AAA (ATPases associated wi th a variety of cellular activities) family member, that co-precipitat es with I kappa B alpha immune complexes, The ubiquitinated I kappa B alpha conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-kappa B. In ultracentrif ugation analysis, physically associated VCP and ubiquitinated I kappa B alpha complexes sediment in the 19 S fractions, while the unmodified I kappa B alpha sediments in the 4.5 S fractions deficient in VCP, Ph osphorylation and ubiquitination of I kappa B alpha are critical for V CP binding, which in turn is necessary but not sufficient for I kappa B alpha degradation; while the N-terminal domain of I kappa B alpha is required in all three reactions, both N- and C-terminal domains are r equired in degradation, Further, VCP co-purifies with the 26 S proteas ome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physi cal and functional link between I kappa B alpha and the 26 S proteasom e and play an important role in the proteasome-mediated degradation of I kappa B alpha.