Nuclear oncoproteins are among the most rapidly degraded intracellular proteins. Previous work has implicated the ubiquitin-mediated proteolytic system in the turnover of short-lived intracellular proteins. In the present study, we have evaluated the potential role of the ubiquitin system in the degradation of the specific nuclear oncoproteins encoded by the N-myc, c-myc, c-fos, p53, and EIA genes. Each of these nuclear oncoproteins was synthesized in vitro by transcription of the appropriate cDNA and translation of the resulting mRNA in the presence of [35SPmethionine. Degradation of labeled proteins was monitored in the ubiquitin cell-free system. ATP stimulated the degradation of all the proteins between 3-and 10-fold. The degradation was completely inhibited by neutralizing antibody directed against the ubiquitinactivating enzyme, El, the first enzyme in the ubiquitinmediated proteolytic cascade. Moreover, degradation in Eldepleted lysates could be restored in each case by the addition of affinity-purifiled El. These data suggest that the ubiquitin system mediates the degradation of these oncoproteins in vitro. Degradation of other proteins, such as superoxide dismutase, cytochrome c, enolase, RNase A, and ornithine decarboxylase, is not mediated by the ubiquitin cell-free system. This suggests that the nuclear oncoproteins studied here possess specific signals that target them for rapid turnover by this proteolytic pathway. Furthermore, the relative sensitivity to degradation ofvarious EIA mutants in vivo is also maintained in the cell-free system, suggesting that the ubiquitin pathway may play a role in the cellular degradation of these proteins as well.The nuclear oncoproteins N-myc, c-myc, c-fos, p53, and ElA are thought to play a role in the regulation of cell growth and differentiation, and they have been implicated in neoplastic transformation. For example, amplification of the N-myc gene has been demonstrated in a subset of neuroblastomas. Amplification of this gene is also correlated with advanced stages of disease, rapid tumor progression, and a poor prognosis (1, 2). c-myc is activated by translocation or mutation in many B-cell lymphomas (3) or by amplification in other tumor types, such as small cell lung cancer and breast cancer (4, 5). The c-fos oncogene product has been implicated in neoplastic transformation as well as in mediating the action of a variety of extracellular stimuli (6, 7). Normal p53 appears to function as a transformation suppressor gene, but mutations have been identified that both stabilize the protein and result in neoplastic transformation (8, 9). Finally, the proteins encoded by early region 1A (EIA) of human adenovirus play a central role in the ability of the virus to replicate efficiently and to transform certain primary cells (10).A feature common to all five of the nuclear oncoproteins described above is their rapid degradation in vivo (11). Rapid degradation of proteins enables the cell to alter the levels of regulatory proteins quickly (12). An interesting...

Degradation of a protein via the ubiquitin system involves two discrete steps, signaling by covalent conjugation of multiple moieties of ubiquitin and degradation of the tagged substrate. Conjugation is catalyzed via a three-step mechanism that involves three distinct enzymes that act successively: E1, E2, and E3. The first two enzymes catalyze activation of ubiquitin and transfer of the activated moiety to E3, respectively. E3, to which the substrate is specifically bound, catalyzes formation of a polyubiquitin chain that is anchored to the targeted protein. The polyubiquitin-tagged protein is degraded by the 26 S proteasome, and free and reutilizable ubiquitin is released. In addition to the three conjugating enzymes, targeting of certain proteins requires association with ancillary proteins and/or post-translational modification(s). Using a specific antibody to deplete cell extract from the molecular chaperone Hsc70, we demonstrate that this protein is required for the degradation of actin, ␣-crystallin, glyceraldehyde-3-phosphate dehydrogenase, ␣-lactalbumin, and histone H2A. In contrast, the degradation of bovine serum albumin, lysozyme, and oxidized RNase A is Hsc70-independent. Mechanistic analysis revealed that the chaperone is required for the conjugation reaction; however, it does not substitute for E3. Involvement of the chaperone in the proteolytic process requires complex formation with the substrate. Formation of this complex appears to be essential in the proteolytic process. In addition, the proper function of the chaperone in the proteolytic process requires the presence of K ؉ , which allows rapid cycles of dissociation and association of the complex. The chaperone may act by binding to the substrate and unfolding it to expose a ubiquitin ligase-binding site. In addition, it can also act directly on the ubiquitination machinery.Degradation of short-lived and key regulatory proteins via the ubiquitin-proteasome pathway plays important roles in basic cellular processes. Protein targets of the ubiquitin system include, among others, cyclins, cyclin-dependent kinases and their inhibitors, tumor suppressors, oncoproteins, and transcriptional activators and their inhibitors. Selection of proteins for degradation can be mediated via primary (constitutive) or secondary signals such as post-translational modifications or via association with ancillary proteins. These signals are recognized by specific ubiquitin-protein ligases (E3), 1 to which the substrate proteins bind prior to ubiquitination. Thus, the ligases play a key role in the ubiquitin proteolytic cascade, recognition and selection of proteins for conjugation and subsequent degradation. Following formation of the polyubiquitin adduct, the protein moiety is degraded by the 26 S proteasome complex, and free and reutilizable ubiquitin is released (reviewed in Refs. 1-4).Molecular chaperones comprise a set of universally conserved proteins that bind and stabilize conformers of other proteins. A regulated, ATP-dependent association-dissociation cyc...

Processing of the p105 precursor to form the active subunit p50 of the NF-kB transcription factor is a unique case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A glycine-rich region along with a downstream acidic domain have been demonstrated to be essential for processing. Here we demonstrate that following IkB kinase (IkK)-mediated phosphorylation, the C-terminal domain of p105 (residues 918±934) serves as a recognition motif for the SCF b-TrCP ubiquitin ligase. Expression of IkKb dramatically increases processing of wild-type p105, but not of p105-D918±934. Dominant-negative b-TrCP inhibits IkK-dependent processing. Furthermore, the ligase and wild-type p105 but not p105-D918±934 associate physically following phosphorylation. In vitro, SCF b-TrCP speci®cally conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IkBs, b-catenin and human immunode®-ciency virus type 1 Vpu. Since p105-D918±934 is also conjugated and processed, it appears that p105 can be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. Keywords: IkB kinase (IkK)/NF-kB/p105/b-TrCP/ ubiquitin IntroductionThe NF-kB transcription factors play key roles in basic processes such as regulation of the immune and in¯am-matory responses, development and differentiation, malignant transformation and apoptosis (Baeuerle and Baltimore, 1996;Baldwin, 1996;Barnes and Karin, 1997;Ghosh et al., 1998;Foo and Nolan, 1999). The precursor molecules p105 and p100 undergo ubiquitinand proteasome-mediated limited processing to yield the respective active subunits p50 and p52 (Palombella et al., 1994;Orian et al., 1995;Betts and Nabel, 1996), which are derived from the N-terminal domain of the molecule. The C-terminal domain is degraded (Fan and Maniatis, 1991). These subunits typically heterodimerize with members of the rel family, such as p65, RelB or c-Rel, to generate the active transcription factor. In the resting cell, the heterodimer generates a ternary complex with a member of the IkB family of inhibitory proteins and is sequestered in the cytosol. Following stimulation, speci®c IkB kinases are activated (Mercurio et al., 1997;Woronicz et al., 1997;Zandi et al., 1997) and phosphorylate the protein on serine residues 32 and 36 (Brown et al., 1995). The phosphorylation leads to recognition of the molecule by the SCF b-TrCP ubiquitin ligase complex (see, for example, Yaron et al., 1998;Winston et al., 1999), polyubiquitylation on Lys21 and/or Lys22 and subsequent degradation by the 26S proteasome (Alkalay et al., 1995;Chen et al., 1995). Following degradation of IkBa, the heterodimer is translocated into the nucleus where it initiates speci®c transcription.The ubiquitin pathway is involved in the regulation of many basic cellular processes, such as cell cycle progression, differentiation and development, and the immune and in¯...

We have previously shown that the degradation of c-myc and N-myc in vitro is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins in vivo and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins