c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination.

The p53 protein is subject to Mdm2-mediated degradation by the ubiquitin-proteasome pathway. This degradation requires interaction between p53 and Mdm2 and the subsequent ubiquitination and nuclear export of p53. Exposure of cells to DNA damage results in the stabilization of the p53 protein in the nucleus. However, the underlying mechanism of this effect is poorly defined. Here we demonstrate a key role for c-Abl in the nuclear accumulation of endogenous p53 in cells exposed to DNA damage. This effect of c-Abl is achieved by preventing the ubiquitination and nuclear export of p53 by Mdm2, or by human papillomavirus E6. c-Abl null cells fail to accumulate p53 efficiently following DNA damage. Reconstitution of these cells with physiological levels of c-Abl is sufficient to promote the normal response of p53 to DNA damage via nuclear retention. Our results help to explain how p53 is accumulated in the nucleus in response to DNA damage.

Mechanisms of ubiquitin-mediated, limited processing of the NF-kappaB1 precursor protein p105.

In most cases, target proteins of the ubiquitin system are completely degraded. In several exceptions, such as the first step in the activation of the transcriptional regulator NF-kappaB, the substrate, the precursor protein p105, is processed in a limited manner to yield the active subunit p50. p50 is derived from the N-terminal domain of p105, whereas the C-terminal domain is degraded. The mechanisms involved in this unique process have remained elusive. We have shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is one important processing signal and that it interferes with processing of the ubiquitinated precursor by the 26S proteasome. Also, amino acid residues 441-454 are important for processing under non-stimulated conditions. Lys 441 and 442 serve as ubiquitination targets, whereas residues 446-454 may serve as a ligase recognition motif. Following IkappaB kinase (IKK)-mediated phosphorylation, the C-terminal domain of p105, residues 918-934, recruits the SCF(beta-TrCP) ubiquitin ligase, and ubiquitination by this complex leads to accelerated processing. The two sites appear to be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. We have shown that ubiquitin conjugation and processing of a series of precursors of p105 that lack the C-terminal IKK phosphorylation/TrCP binding domain, is progressively inhibited with increasing number of ankyrin repeats. Inhibition is due to docking of active NF-kappaB subunits to the ankyrin repeat domain in the C-terminal half of p105 (IkappaBgamma). Inhibition is alleviated by phosphorylation of the C-terminal domain that leads to ubiquitin-mediated degradation of the ankyrin repeat domain and release of the anchored subunits. We propose a model that may explain the requirement for two sites: a) a basal site that may be involved in co-translational processing prior to the synthesis of the ankyrin repeat domain; and b) a signal-induced site that is involved in processing/degradation of the complete molecule following cell activation, with rapid release of stored, transcriptionally active subunits.

SCF(beta)(-TrCP) ubiquitin ligase-mediated processing of NF-kappaB p105 requires phosphorylation of its C-terminus by IkappaB kinase.

Processing of the p105 precursor to form the active subunit p50 of the NF-kappaB 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 IkappaB kinase (IkappaK)-mediated phosphorylation, the C-terminal domain of p105 (residues 918-934) serves as a recognition motif for the SCF(beta)(-TrCP) ubiquitin ligase. Expression of IkappaKbeta dramatically increases processing of wild-type p105, but not of p105-Delta918-934. Dominant-negative beta-TrCP inhibits IkappaK-dependent processing. Furthermore, the ligase and wild-type p105 but not p105-Delta918-934 associate physically following phosphorylation. In vitro, SCF(beta)(-TrCP) specifically conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IkappaBs, beta-catenin and human immunodeficiency virus type 1 Vpu. Since p105-Delta918-934 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.

Identification of the ubiquitin carrier proteins, E2s, involved in signal-induced conjugation and subsequent degradation of IkappaBalpha.

The last step in the activation of the transcription factor NF-kappaB is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor IkappaBalpha. Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive. Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process. The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of IkappaBalpha that cannot be phosphorylated and conjugated following an extracellular signal. Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor. Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor alpha-induced degradation of the inhibitor in vivo. Not surprisingly, they have a similar effect in a cell-free system as well. Although it is clear that the E2 enzymes are not entirely specific to IkappaBalpha, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. The mechanisms that underlie the involvement of two different E2 species in IkappaBalpha conjugation are not clear at present. It is possible that different conjugating machineries operate under different physiological conditions or in different cells.

Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway.

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 in cells is inhibited by lactacystin and MG132, two inhibitors of the 20S proteasome. Inhibition is accompanied by accumulation of myc-ubiquitin conjugates. Dissection of the ancillary proteins involved revealed that the high-risk human papillomavirus oncoprotein E6-16 stimulates conjugation and subsequent degradation of the myc proteins in vitro. Expression of E6-16 in cells results in significant shortening of the t1/2 of the myc proteins with subsequent decrease in their cellular level. Analysis of the conjugating enzymes revealed that under basal conditions the proteins can be conjugated by two pairs of E2s and E3s-E2-14 kDa and E3alpha involved in the "N-end rule" pathway, and E2-F1 (UbcH7) and E3-Fos involved also in conjugation of c-Fos. In the presence of E6-16, a third pair, E2-F1 and E6-AP mediate conjugation of myc by means of a mechanism that appears to be similar to that involved in the targeting of p53, formation of a myc. E6.E6-AP targeting complex. It is possible that in certain cells E6-mediated targeting of myc prevents myc-induced apoptosis and thus ensures maintenance of viral infection.

Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro differentiation and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells.

By means of differential RNA display, we have isolated a cDNA corresponding to transcripts that are down-regulated upon differentiation of the goblet cell-like HT-29-M6 human colon carcinoma cell line. These transcripts encode proteins originally identified as CROC-1 on the basis of their capacity to activate transcription of c-fos. We show that these proteins are similar in sequence, and in predicted secondary and tertiary structure, to the ubiquitin-conjugating enzymes, also known as E2. Despite the similarities, these proteins lack a critical cysteine residue essential for the catalytic activity of E2 enzymes and, in vitro, they do not conjugate or transfer ubiquitin to protein substrates. These proteins constitute a distinct subfamily within the E2 protein family and are highly conserved in phylogeny from yeasts to mammals. Therefore, we have designated them UEV (ubiquitin-conjugating E2 enzyme variant) proteins, defined as proteins similar in sequence and structure to the E2 ubiquitin-conjugating enzymes but lacking their enzymatic activity (HW/GDB-approved gene symbol, UBE2V). At least two human genes code for UEV proteins, and one of them, located on chromosome 20q13.2, is expressed as at least four isoforms, generated by alternative splicing. All human cell types analyzed expressed at least one of these isoforms. Constitutive expression of exogenous human UEV in HT-29-M6 cells inhibited their capacity to differentiate upon confluence and caused both the entry of a larger proportion of cells in the division cycle and an accumulation in G2-M. This was accompanied with a profound inhibition of the mitotic kinase, cdk1. These results suggest that UEV proteins are involved in the control of differentiation and could exert their effects by altering cell cycle distribution.

Ubiquitin-dependent degradation of certain protein substrates in vitro requires the molecular chaperone Hsc70.

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, alpha-crystallin, glyceraldehyde-3-phosphate dehydrogenase, alpha-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.

Isolation, characterization, and partial purification of a novel ubiquitin-protein ligase, E3. Targeting of protein substrates via multiple and distinct recognition signals and conjugating enzymes.

Degradation of a protein via the ubiquitin system involves two discrete steps, conjugation of ubiquitin to the substrate and degradation of the adduct. Conjugation follows a three-step mechanism. First, ubiquitin is activated by the ubiquitin-activating enzyme, E1. Following activation, one of several E2 enzymes (ubiquitin-carrier proteins or ubiquitin-conjugating enzymes, UBCs) transfers ubiquitin from E1 to the protein substrate that is bound to one of several ubiquitin-protein ligases, E3s. These enzymes catalyze the last step in the process, covalent attachment of ubiquitin to the protein substrate. The binding of the substrate to E3 is specific and implies that E3s play a major role in recognition and selection of proteins for conjugation and subsequent degradation. So far, only a few ligases have been identified, and it is clear that many more have not been discovered yet. Here, we describe a novel ligase that is involved in the conjugation and degradation of non "N-end rule" protein substrates such as actin, troponin T, and MyoD. This substrate specificity suggests that the enzyme may be involved in degradation of muscle proteins. The ligase acts in concert with E2-F1, a previously described non N-end rule UBC. Interestingly, it is also involved in targeting lysozyme, a bona fide N-end substrate that is recognized by E3 alpha and E2-14 kDa. The novel ligase recognizes lysozyme via a signal(s) that is distinct from the N-terminal residue of the protein. Thus, it appears that certain proteins can be targeted via multiple recognition motifs and distinct pairs of conjugating enzymes. We have purified the ligase approximately 200-fold and demonstrated that it is different from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. The native enzyme has an apparent molecular mass of approximately 550 kDa and appears to be a homodimer. Because of its unusual size, we designated this novel ligase E3L (large). E3L contains an -SH group that is essential for its activity. Like several recently described E3 enzymes, including E6-AP and the ligase involved in the processing of p105, the NF-kappa B precursor, the novel ligase is found in mammalian tissues but not in wheat germ.

Degradation of ornithine decarboxylase by the mammalian and yeast 26S proteasome complexes requires all the components of the protease.

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is an extremely short-lived protein. This attribute is important for the regulation of the activity of the enzyme and implies that the mechanisms involved in its degradation play an important role in the control of the cellular processes in which the enzyme is involved. Recently, it has been shown that ODC is degraded by the 26S proteasome complex in a process that requires antizyme, but not ubiquitin. With one reported exception, ODC, the 26S complex recognizes and degrades specifically ubiquitinated proteins. Their unconjugated counterparts are not targeted. The 26S complex is composed of a core catalytic unit, the 20S proteasome complex, and two additional, and apparently distinct, subcomplexes. The two additional subcomplexes are regulatory subunits that are required in order to confer specificity and control. In this study, we demonstrate that, like the degradation of ubiquitin-conjugated proteins, ubiquitin-independent degradation of ODC also requires prior assembly of the mammalian 26S proteasome from all the three subunits, the 20S proteasome and the two subcomplexes. The combination of any two subunits does not support generation of a proteolytically active complex. This is also true for the yeast 26S complex. Like the mammalian 20S proteasome, the yeast 20S complex can cleave short peptides in an ATP-independent mode, but cannot degrade ODC or ubiquitin-conjugated proteins. These proteins are degraded only following addition of the regulatory subunits and generation of the high-molecular-mass 26S complex. In a distinct, but related, set of experiments, we demonstrate that the degradation of ODC by the assembled 26S proteasome in vitro reproduces faithfully proteolysis of the enzyme in the intact cell. Namely, (a) a C-terminal-deleted mouse ODC and trypanosome ODC are stable both in vitro and in vivo, and (b) like proteolysis in the intact cell, degradation in the reconstituted cell-free system is also dependent upon the addition of antizyme.

Complete reconstitution of conjugation and subsequent degradation of the tumor suppressor protein p53 by purified components of the ubiquitin proteolytic system.

The wild-type tumor suppressor protein p53 is a short-lived protein that plays important roles in regulation of cell cycle, differentiation, and survival. Mutations that inactivate or alter the tumor suppressor activity of the protein seem to be the most common genetic change in human cancer and are frequently associated with changes in its stability. The ubiquitin system has been implicated in the degradation of p53 both in vivo and in vitro. A mutant cell line that harbors a thermolabile ubiquitin-activating enzyme, E1, fails to degrade p53 at the nonpermissive temperature. Studies in cell-free extracts have shown that covalent attachment of ubiquitin to the protein requires the three conjugating enzymes: E1, a novel species of ubiquitin-carrier protein (ubiquitin-conjugating enzyme; UBC),E2-F1, and an ubiquitin-protein ligase, E3. Recognition of p53 by the ligase is facilitated by formation of a complex between the protein and the human papillomavirus (HPV) oncoprotein E6. Therefore, the ligase has been designated E6-associated protein (E6-AP). However, these in vitro studies have not demonstrated that the conjugates serve as essential intermediates in the proteolytic process. In fact, in many cases, conjugation of ubiquitin to the target protein does not signal its degradation. Thus, it is essential to demonstrate that p53-ubiquitin adducts serve as essential proteolytic intermediates and are recognized and degraded by the 26S protease complex, the proteolytic arm of the ubiquitin pathway. In this study, we demonstrate that conjugates of p53 generated in the presence of purified, E1, E2, E6-AP, E6, ubiquitin and ATP, are specifically recognized by the 26S protease complex and degraded. In contrast, unconjugated p53 remains stable. The ability to reconstitute the system from purified components will enable detailed analysis of the recognition process and the structural motifs involved in targeting the protein for degradation.

Degradation of the tumor suppressor protein p53 by the ubiquitin-mediated proteolytic system requires a novel species of ubiquitin-carrier protein, E2.

The tumor suppressor protein p53 is extremely unstable in most cell lines. In contrast, many mutant and oncogenic species of the protein are stable. The degradation of p53 in vivo requires metabolic energy; however, the proteolytic system(s) involved have not been identified. The ubiquitin system has been implicated in the degradation of p53 in vitro. The degradation is stimulated significantly by the human papillomavirus (HPV) oncoprotein E6 that associates with p53 and facilitates conjugate formation and subsequent degradation. Complex formation between E6 and p53 is promoted by a cellular protein designated E6-associated protein (E6-AP). Initial dissection of the conjugation process have demonstrated a role for the ubiquitin-activating enzyme, E1, but the ubiquitin-carrier protein (E2, UBC) and the ubiquitin protein ligase, E3, have not been identified. In this study, we report that a novel species of ubiquitin-carrier protein designated E2-F1 (Blumenfeld, N., Gonen, H., Mayer, A., Smith, C., Siegel, N.R., Schwartz, A.L., and Ciechanover, A. (1994) J. Biol. Chem. 269, 9574-9581) is involved in the conjugation and degradation of p53. This E2 enzyme recognizes non-"N-end rule" protein substrates and appears to mediate their conjugation via a novel species of E3. The process of recognition appears to be selective; E2-F1 is not required for the conjugation and degradation of human N-myc. The involvement of E2-F1 in the in vitro process appears to be physiologically meaningful and to reproduce the in vivo process; mutant species of p53 that do not interact with E6 and are stable in vivo are not recognized by the cell free system.

Degradation of nuclear oncoproteins by the ubiquitin system in vitro.

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 E1A 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 [35S]methionine. 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 ubiquitin-activating enzyme, E1, the first enzyme in the ubiquitin-mediated proteolytic cascade. Moreover, degradation in E1-depleted lysates could be restored in each case by the addition of affinity-purified E1. 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 of various E1A 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.