Involvement of valosin-containing protein, an ATPase co-purified with IκBα and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IκBα

The inactivation of the prototype NF-kappa B inhibitor, I kappa B alpha, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates 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 ultracentrifugation 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, Phosphorylation and ubiquitination of I kappa B alpha are critical for VCP 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 required in degradation, Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and functional link between I kappa B alpha and the 26 S proteasome and play an important role in the proteasome-mediated degradation of I kappa B alpha.