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-B inhibitor, IB␣, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosincontaining protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates with IB␣ immune complexes. The ubiquitinated IB␣ conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-B. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IB␣ complexes sediment in the 19 S fractions, while the unmodified IB␣ sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IB␣ are critical for VCP binding, which in turn is necessary but not sufficient for IB␣ degradation; while the N-terminal domain of IB␣ 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 IB␣ and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IB␣.Transcription factor NF-B is involved in a large variety of processes, such as cell growth, transcriptional regulation, immune, inflammatory, and stress responses (reviewed in Refs. 1-4). NF-B is a homo-or heterodimer consisting of various combinations of the family members, including NFB1 (p50 and precursor p105), c-Rel, RelA, NFB2 (p52 and precursor p100), RelB, and Drosophila proteins Dorsal and Dif. Unlike many other transcription factors that are localized in the nucleus, the NF-B dimeric factor is sequestered in the cytoplasm of most cells through binding to a family of inhibitor proteins, IB. In response to extracellular stimuli, the inhibitors are partially or entirely degraded, thus liberating the DNA-binding dimer for translocation to the nucleus. The I6B family contains IB␣, IB␤, IB␥, Bcl-3, the precursor proteins p105 and p100, and the Drosophila protein Cactus. All members of the IB family contain 5-8 ankyrin motifs, thought to be involved in protein-protein interactions. It has been shown that when the precursor protein p105 is involved as the inhibitor, the processing from p105 to the active p50 occurs through the ubiquitindependent proteasome (Ub-Pr) 1 pathway, which degrades the C-terminal ankyrin-containing domain of p105 (5, 6).2 For the prototype complex that contains p50, p65, and IB␣, upon stimulation the entire NF-B complex becomes hyperphosphorylated. The induced phosphorylation of IB␣ does not lead to its immediate dissociation from the complex; rather, it signals for rapid IB␣ degradation, thus liberating the active p50⅐p65 dimer for translocation to the nucleus (7-15). We and others showed that the degradation of IB␣ is sensitive to proteasome inhibitors and is ubiquitin-dependent. Recently, it was further shown that signal-ind...

Pre-B-Cell Leukemia Transcription Factor 1 Regulates Expression of Valosin-Containing Protein, a Gene Involved in Cancer Growth

“…16 VCP is involved in NF-B activation, which works in an anti-apoptotic function. 6,7 Dysfunction of VCP results in vacuole and inclusion body formation, ultimately leading to cell death. 17 Mutated VCP causes inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia.…”

“…[1][2][3][4][5] Notably, VCP is involved in activation of nuclear factor (NF)-B, a transcription factor promoting antiapoptosis, cell proliferation, and invasion. 6 VCP is abundant in all kinds of cells, accounting for more than 1% of total cellular protein. 1 Recently, we showed that VCP is associated with metastatic potential of murine osteosarcoma cell line by using mRNA subtraction technique: the cell lines transfected with VCP showed the constant activation of NF-B, decreased apoptosis rates after stimulation with tumor necrosis factor (TNF)-␣, and increased metastatic potential.…”

The heavy metal cadmium induces valosin-containing protein (VCP)-mediated aggresome formation

“…The cell extracts were cleared by centrifugation (12000 × g, 10 min) and immunoprecipitated with an anti-GFP antibody (Sigma-Aldrich, St. Louis, MO). Protein samples were electrophoresed using SDS-PAGE, transferred to nitrocellulose membranes, then analyzed by immunoblotting with anti-VCP or HDAC6 antibodies and HRP-labeled secondary antibodies (Dai et al, 1998;Dai and Li, 2001b).…”

Distinct Roles for the AAA ATPases NSF and p97 in the Secretory Pathway

“…These include roles in ubiquitin-and proteasome-dependent degradation of cytosolic proteins (Ghislain et al, 1996;Dai et al, 1998;Dai and Li, 2001), ER-associated degradation (ERAD; reviewed in Bays and Hampton, 2002;Tsai et al, 2002), and regulated ubiquitindependent processing (Rape et al, 2001 1997), Ufd1/Npl4 (Meyer et al, 2000(Meyer et al, , 2002, VCIP135 (Uchiyama et al, 2002), and SVIP (Nagahama et al, 2003). Although the mechanism by which AAA ATPases such as NSF and p97 use ATP to modulate the structure of their substrates is not known, the overall homology between these two enzymes makes the idea that they might operate by a similar mechanism attractive.…”

“…These include roles in ubiquitin-and proteasome-dependent degradation of cytosolic proteins (Ghislain et al, 1996;Dai et al, 1998;Dai and Li, 2001), ER-associated degradation (ERAD; reviewed in Bays and Hampton, 2002;Tsai et al, 2002), and regulated ubiquitindependent processing (Rape et al, 2001). It is believed that different adaptor proteins direct p97 to perform these varied cellular activities.…”