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

Abstract

The inactivation of the prototype NF-κB inhibitor, IκBα, 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 IκBα immune complexes. The ubiquitinated IκBα conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-κB. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IκBα complexes sediment in the 19 S fractions, while the unmodified IκBα sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IκBα are critical for VCP binding, which in turn is necessary but not sufficient for IκBα degradation; while the N-terminal domain of IκBα 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 IκBα and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IκBα.