Ubiquitin-mediated Processing of NF-κB Transcriptional Activator Precursor p105

Abstract

In most cases, the transcriptional factor NF-B is a heterodimer consisting of two subunits, p50 and p65, which are encoded by two distinct genes of the Rel family. p50 is translated as a precursor of 105 kDa. The C-terminal domain of the precursor is rapidly degraded, forming the mature p50 subunit consisted of the N-terminal region of the molecule. The mechanism of generation of p50 is not known. It has been suggested that the ubiquitin-proteasome system is involved in the process; however, the specific enzymes involved and the mechanism of limited proteolysis, in which half of the molecule is spared, have been obscure. Palombella and colleagues (Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) Cell 78, 773-785) have shown that ubiquitin is required for the processing in a cell-free system of a truncated, artificially constructed, 60-kDa precursor. They have also shown that proteasome inhibitors block the processing both in vitro and in vivo. In this study, we demonstrate reconstitution of a cell-free processing system and demonstrate directly that: (a) the ubiquitin-proteasome system is involved in processing of the intact p105 precursor, (b) conjugation of ubiquitin to the precursor is an essential intermediate step in the processing, (c) the recently discovered novel species of the ubiquitin-carrier protein, E2-F1, that is involved in the conjugation and degradation of p53, is also required for the limited processing of the p105 precursor, and (d) a novel, ϳ320-kDa species of ubiquitin-protein ligase, is involved in the process. This novel enzyme is distinct from E6-AP, the p53-conjugating ligase, and from E3␣, the "N-end rule" ligase.NF-B and other members of the Rel family of transcriptional activators have recently gained considerable attention because of their unique mechanism of activation, and their active role in cytoplasmic-nuclear signaling in response to a variety of extracellular stimuli. In most cases, NF-B is a heterodimeric complex composed of p50 and p65 subunits. p50 is initially synthesized as an inactive 105-kDa precursor that is processed to the mature protein following removal of the Cterminal domain of the molecule. A second precursor molecule, p100, gives rise to the p52 DNA-binding subunit of NF-B, probably via a similar mechanism. The heterodimeric complex was originally identified as an inducible B cell-specific transcription factor able to bind the light chain enhancer (1). NF-B is now recognized to be a ubiquitously expressed factor that is present as an inactive ternary complex in the cytosol of most cells. In this ternary complex, a third inhibitory protein belonging to the IB family is associated with the heterodimer (for recent reviews, see Refs. 2-4). A wide range of stimuli such as bacterial products, parasites, viruses and viral products, inflammatory cytokines, T cells, B cells, fibroblast mitogens, protein synthesis inhibitors, physical and oxidative stresses, and drugs lead to accelerated processing of the precursor, degradation of the inhibitor, ...

Ubiquitin-dependent Degradation of Certain Protein Substrates in Vitro Requires the Molecular Chaperone Hsc70

“…7 (lanes 1 and 2), addition of E1, E2-F1 (or UbcH5; data not shown), and the chaperone to a cell-free reconstituted system is not sufficient to restore conjugation. Conjugate formation is dependent upon the addition of Fraction IIA (lanes 3 and 4), which contains all the known E3 enzymes (34,36,43). Depletion of Hsc70 from Fraction IIA inhibits conjugation (lane 5), whereas, as expected, addition of purified Hsc70 to the system restores activity.…”

“…E2-F1 was purified to homogeneity from Fraction I, and Fraction IIA was prepared from Fraction II by (NH 4 ) 2 SO 4 precipitation (0 -38%) as described (34). UbcH5 cDNA (35) was expressed in E. coli BL21 cells, and the enzyme was purified from the bacterial extracts following induction as described (36).…”

Mechanism of processing of the NF-κB2 p100 precursor: identification of the specific polyubiquitin chain-anchoring lysine residue and analysis of the role of NEDD8-modification on the SCFβ-TrCP ubiquitin ligase

“…There are several levels of regulation of NF-kB. The first involves ubiquitin-mediated generation of the mature p50 and p52 subunits from their respective larger precursors p105 and p100 (Fan and Maniatis, 1991;Palombella et al, 1994;Orian et al, 1995;Fong and Sun, 2002).…”

Regulation of apoptosis proteins in cancer cells by ubiquitin

“…Over the past few years, apoptosis-pathway molecules have been identified as substrates and targets for proteasome degradation leading to apoptosis resistance. Apoptosis molecules that are regulated by the ubiquitin-proteasome pathway include members of the Bcl-2 family (Chang et al, 1998;Breitschopf et al, 2000;Li and Dou, 2000), IAP family (Yang and Li, 2000;Suzuki et al, 2001;MacFarlane et al, 2002;Martin, 2002) and inhibitor of the kB (IkB) (Orian et al, 1995;Jesenberger and Jentsch, 2002;Kovalenko et al, 2003). Alteration of the stability of these proteins through the ubiquitin-proteasome-regulated pathway generally contributes to apoptosis resistance in cancer cells.…”

“…The selective degradation or stabilization of intracellular proteins by ubiquitin-dependent pathways is essential for correcting the regulation of many cellular processes. Ubiquitin is covalently attached to substrate proteins by a protein complex usually including an activating enzyme (E1), a conjugating enzyme (E2) and a protein ligase (E3) (Orian et al, 1995;Sudakin et al, 1995;Haas and Siepmann, 1997). The addition of four or more ubiquitin moieties targets the substrates for destruction via the 26S proteasome, and free ubiquitin is recycled.…”