Degradation of E2A proteins through a ubiquitin-conjugating enzyme, UbcE2A.

The helix-loop-helix E2A proteins (E12 and E47) govern cellular growth and differentiation. To identify binding partners that regulate the function of these ubiquitous transcription factors, we screened for proteins that interacted with the C terminus of E12 by the yeast interaction trap. UbcE2A, a rat enzyme that is highly homologous to and functionally complements the yeast ubiquitin-conjugating enzyme UBC9, was identified and cloned. UbcE2A appears to be an E2A-selective ubiquitin-conjugating enzyme because it interacts specifically with a 54-amino acid region in E47-(477-530) distinct from the helix-loop-helix domain. In contrast, most of the UbcE2A protein is required for interaction with an E2A protein. The E2A proteins appear to be degraded by the ubiquitin-proteasome pathway because the E12 half-life of 60 min is extended by the proteasome inhibitor MG132, and E12 is multi-ubiquitinated in vivo Finally, antisense UbcE2A reduces E12 degradation. By participating in the degradation of the E2A proteins, UbcE2A may regulate cell growth and differentiation.

involved in neuronal differentiation (5), and SCL/TAL, which is involved in hematopoiesis (6). The E2A proteins also form homodimers that are linked by intermolecular disulfide bonds in B cells but not muscle cells (7). These homodimers are thought to be the predominant DNA-binding species in B cells (8). In mice carrying a null mutation in E2A, immunoglobulin gene segments do not rearrange and the animals lack mature B lymphocytes (9,10).
In addition to its role in cellular differentiation, the E2A gene is the breakpoint of two translocations associated with childhood lymphoid leukemia. A truncated E2A gene fuses to the PBX1 homeobox gene (11) and to the HLF basic leucine zipper gene (12). Because the E2A portion is required for transformation in both instances, E2A proteins appear to play a role in growth control. Peverali et al. (13) have shown that overexpression of E12 or E47 inhibits cell proliferation and mediates arrest of growth a few hours before the G 1 -S transition of the cell cycle. The level of E2A proteins at different stages of the cell cycle could also determine whether cells proliferate or differentiate.
Certain transcription factors and cell cycle regulators are degraded rapidly in vivo (14). For example, c-Fos and c-Jun, which have half-lives of about 30 and 90 min, respectively, cause uncontrolled cell proliferation if their expression goes unchecked (15,16). Also, the cyclins and cyclin-dependent kinase inhibitors must undergo programmed destruction if the cell cycle is to continue (17,18). The ubiquitin-proteasome pathway fosters the rapid turnover of many cell regulators. These include the transcription factors MAT␣2 (19) and GCN4 (20) from yeast, c-Fos (21) and c-Jun (22), and the cell cycle regulators cyclin B (23) and cyclin-dependent kinase inhibitor p27 (24). The ubiquitin-proteasome pathway also mediates processing of the p105 precursor of NF-B and degradation of its inhibitor protein IB␣ (25,26).
The ubiquitin-proteasome pathway involves covalent conjugation of a target protein to ubiquitin molecules, degradation of that protein, and release of reusable ubiquitin, as reviewed by Ciechanover (27). Ubiquitin is activated initially by ATP in a reaction catalyzed by the enzyme E1. The activated ubiquitin is then transferred to a ubiquitin-conjugating enzyme, E2, that catalyzes formation of an isopeptide bond between the C-terminal glycine of ubiquitin and the ⑀-amino group of a lysine residue on the target protein. For many proteins, conjugation to ubiquitin also requires a specific ubiquitin-protein ligase, E3. A mono-ubiquitinated target protein then undergoes further ubiquitination to produce multi-ubiquitinated chains (28). These ubiquitin conjugates are recognized by a multisubunit regulatory complex on the proteasome that also unfolds and translocates them to the barrel-shaped 20 S core, where they are degraded (29,30).
Despite the importance of the E2A proteins in cell growth and differentiation, little is known about the mechanisms regulating their stability. While isolating proteins that bound to E12 by the yeast interaction trap, we cloned UbcE2A, the rat homologue of the yeast ubiquitin-conjugating enzyme UBC9. We found that E12 turns over rapidly and is multi-ubiquitinated and that its half-life is extended by a proteasome inhibitor. Moreover, antisense UbcE2A reduces E12 degradation. These observations suggest that E12 is regulated by the ubiquitin-proteosome pathway. By regulating the level of E12, UbcE2A may regulate the cell cycle.

EXPERIMENTAL PROCEDURES
Plasmids-Escherichia coli and nucleic acids were manipulated as described by Ausubel et al. (31). We were given the following cDNAs: E12 and E47 (11), deletions and point mutants of E47 generated by PCR (13), and mouse c-Myc (32). We cloned the following cDNAs by reverse transcriptase PCR and confirmed their sequences: rat Id3 (33): rat Max (34), human OCT-1 (35), and rat c-Jun (36). Mathias Treier (European Molecular Biology Laboratory, Heidelberg, Germany) provided the ubiquitin construct pCMVHA-Ub (22). The pCR 3 vector (Invitrogen) containing the cytomegalovirus enhancer and promoter and a bovine growth hormone polyadenylation signal was used for expression in eukaryotic cells. Full-length E12, UbcE2A, and c-Jun cDNAs were amplified by PCR and ligated into pCR 3 by TA cloning. cDNA authenticity was confirmed by dideoxy sequencing and translation of the appropriate protein in vitro. Various E12, E47, and UbcE2A deletion mutants were generated by standard PCR techniques and sequenced. Hemagglutinin (HA)-tagged UbcE2A contained the sequence MASYPY-DVPDYASPEF added to the N terminus of full-length UbcE2A. The pGEX4T vector (Pharmacia Biotech Inc.) was used to express glutathione S-transferase (GST) fusion proteins in E. coli.
Yeast Interaction Trap Experiments-We screened for E12-interacting proteins by the yeast two-hybrid interaction trap according to Gyuris et al. (37). EGY48 (MAT␣ trp1 ura3 his3 LEU2::pLexop6-LEU2) was used as host yeast strain in all interaction experiments. All bait plasmids were constructed by inserting the corresponding cDNA (inframe) downstream of the lexA gene contained in pEG202 (38). The oligo(dT)-primed rat aortic cDNA library used for screening had been constructed with the yeast galactose-inducible expression plasmid pJG4-5 (37). This library comprises 4.5 ϫ 10 6 members, 88% of which contain a cDNA insert whose average size ranges between 0.6 and 2.3 kilobase pairs. We began the interaction screen with an EGY48-p1840-pLexA-E12-(477-654) (amino acids 477-654 of human E12) strain. pLexA-E12-(477-654) did not spontaneously activate transcription of either reporter gene (lacZ or LEU2) used in this system. We confirmed expression of the appropriate bait protein by Western blotting with both anti-LexA antibody (gift of Barak Cohen, Massachusetts General Hospital, Boston) and anti-E12/E47 antibody. We introduced the rat aortic cDNA library into the EGY48-p1840-pLexA-E12-(477-654) strain according to the procedure of Gietz et al. (39), with modification. A total of 4 ϫ 10 6 transformants was obtained. Plasmids were screened and recovered as described by Gyuris et al. (37). Library plasmids were classified by their restriction patterns after digestion with EcoRI and XhoI and either HinfI or HaeIII. Plasmid DNAs from each class were retested in the interaction-trap assay with pEG202 and pLexA-E12-(477-654). Galactose-inducible expression of an HA-tagged fusion protein in the transformants was also confirmed with the anti-HA antibody 12CA5.
To assess the specificity of interaction and map the interaction domains, we transformed yeast of the EGY48/pSH18-34 strain with the library/interactant plasmids and the bait constructs indicated in Fig. 3 and applied them to glucose ura-his-trp-plates. The bait constructs used in the specificity test were LexA-Id3 (which contains all of the rat Id3 coding sequence), LexA-c-Myc (which contains the C-terminal 137 amino acids of mouse c-Myc), LexA-Max (which contains all of the rat Max coding sequences), and LexA-OCT-1 (amino acids 294 -429 of human OCT-1 containing the POU domain). Eight to twelve colonies from each bait/interactant combination were picked and applied in duplicate to ura-his-trp-plates containing 5-bromo-4-chloro-3-indolyl-␤-D-galactoside, and either 2% glucose or 2% galactose, and 1% raffinose. We checked the color of the yeast 48 h later.
Crude extracts were assayed for yeast ␤-galactosidase activity as described by Kaiser et al. (40). Cells bearing the appropriate bait and interaction plasmids were grown to saturation (overnight at 30°C) in minimal ura-his-trp-medium with 2% glucose. The next day, cells were diluted 1:50 into medium containing 2% galactose and 1% raffinose and allowed to grow overnight. Lysates were then prepared and permeabilized as described (41). Cell concentrations were determined by measuring absorbance at 600 nm. ␤-Galactosidase units were calculated by the equation 1000(A 420 )/(time(min)⅐vol(ml)⅐A 600 ).
Transfection and Immunofluorescence-NIH3T3 fibroblasts were transfected by the calcium phosphate method. Colonies were picked with cloning cylinders after 18 -21 days and expanded. Integration of transfected DNA in the transformants was confirmed by Southern blot analysis. COS7 cells were transfected transiently by electroporation. For immunofluorescence studies, transfected COS7 cells were grown to 75% confluence on chamber slides (Nunc). Cells were washed once with PBS and fixed for 20 min in 2% sucrose with 4% paraformaldehyde at room temperature. Fixed and permeabilized cells were hydrated in PBS for 5 min and incubated with 10% nonimmune rabbit serum in PBS with 0.1% Triton X-100 at room temperature for 20 min to suppress nonspecific binding of IgG. The slides were stained with anti-HA antibody 12CA5 (1:400 dilution) in a moist chamber for 1 h at room temperature. After three washes in PBS with 0.1% Triton X-100, the slides were incubated with 250 l of rhodamine-conjugated goat anti-mouse IgG diluted 1:200 for 45 min at room temperature. The slides were washed extensively again and counterstained with Hoechst 33258 for 5 min, mounted, and viewed in a Nikon fluorescence microscope. 12CA5 staining and Hoechst staining were visualized and photographed in the same fields by changing filter sets.
In Vitro Binding Assays-GST fusion proteins were expressed and purified essentially as described by Smith and Johnson (42). Fresh, overnight cultures of E. coli (HB101) transformed with pGEX4T or pGEX4T E12-(477-654) were diluted 1:10 in LB medium containing ampicillin (100 g/ml) and incubated (with shaking) for 3-5 h at 37°C until the A 600 reached 0.8. Isopropyl ␤-D-thiogalactopyranoside was then added to a final concentration of 0.4 mM, and incubation was allowed to continue for another 3 h. Bacterial cultures were pelleted and resuspended in PBS with 1 mM phenylmethylsulfonyl fluoride and 1% (v/v) aprotinin. The bacteria were then lysed on ice by mild sonication, mixed with Triton X-100 to a final concentration of 1%, and centrifuged at 14,000 ϫ g for 5 min at 4°C. Aliquots (1 ml) of bacterial supernatant were rocked for 30 min at 4°C with 25 l of glutathione-Sepharose 4B (Pharmacia). The Sepharose beads were then washed three times with PBS. 35 S-Labeled proteins were generated by using the TNT T7-coupled reticulocyte lysate system (Promega) with the Id3 or UbcE2A expression construct in pCite4 (Novagen). 35 S-Labeled protein (3 l) was incubated with the beads (25 l) in 50 mM NaCl and bovine serum albumin (1 mg/ml) at 4°C for 1 h (43). The beads were then washed four times with 0.1% Nonidet P-40 in PBS. Protein on the beads was fractionated by SDS-PAGE, stained with Coomassie Blue, and exposed to Kodak x-ray film.
Pulse-Chase Experiments and Immunoprecipitation-COS7 cells in 100-mm dishes (at about 80% confluence) were starved in Met-free DMEM (supplemented with 5% dialyzed fetal bovine serum) for 60 min at 37°C. Cells were then pulse-labeled at 37°C with 100 Ci/ml [ 35 S]Met for 60 min at 37°C. Cells were chased in warm DMEM supplemented with 100 g/ml Met. For the experiment with the proteasome inhibitor MG132 (a gift from Alfred Goldberg, Harvard Medical School, Boston, MA), the inhibitor (at a concentration of 50 M) was added 1 h before pulse-chase and was present throughout the pulsechase periods. After the chase, dishes were washed three times with PBS and then lysed with 3 ml of ice-cold RIPA (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and Complete protease inhibitor mixture (Boehringer Mannheim)) for 20 min at 4°C. Lysates were cleared of nuclei and debris by centrifugation at 14,000 ϫ g at 4°C for 15 min. Spun samples were then cleared with normal mouse serum and protein G-agarose (Pierce) for 1 h at 4°C. 35 S incorporation in the total protein pool was determined by trichloroacetic acid precipitation. Lysate volumes were adjusted so that each extract contained equivalent amounts of radioactivity (trichloroacetic acid-precipitable counts/min).
For E12 immunoprecipitation, lysates were incubated overnight at 4°C with 1-2 g of purified anti-E12/E47 antibody and immobilized protein G. E12 bound to the beads was washed four times with RIPA and subjected to SDS-PAGE followed by fluorography. E12 reactivity in the bands was measured on a PhosphorImager (Molecular Dynamics).
In Vivo Ubiquitination Assay-COS7 cells were electroporated with 6 g of the E12 or c-Jun expression construct and 20 g of the HAtagged ubiquitin expression vector. After 48 h, cells were lysed on ice in RIPA buffer with 10 mM N-ethylmaleimide. The cells were harvested, and cysteine was added to a final concentration of 0.1% to inactivate the N-ethylmaleimide. Cell extracts were immunoprecipitated as described for the pulse-chase experiments; proteins were separated by 10% SDS-PAGE and blotted onto Immobilon-P membranes (Millipore). Blots were immunostained successively with anti-HA antibody 12CA5 and anti-E12 antibody. Reactive products were visualized with an ECL kit (Amersham Corp.).

RESULTS
A Ubiquitin-conjugating Enzyme Cloned by the Yeast Interaction Trap-We used the yeast interaction trap cloning system (37) to identify proteins that interact with the C terminus of E12. A bait expression vector was constructed by fusing the LexA-binding domain to the C terminus of E12 (amino acids 477-654), which includes the basic and HLH domains. We screened a rat aorta cDNA expression library with LexA-E12-(477-654) and identified 42 positive clones from 3.5 ϫ 10 6 transformants. Of the 42 positive clones, 29 encoded Id3 (33) and five encoded Id1 (44), indicating that specific protein-protein interactions were detectable in yeast with our E12 construct. Of the eight remaining clones, five encoded a ubiquitinconjugating enzyme containing the highly conserved active site. We named this gene ubcE2A.
A comparison of the predicted amino acid sequence of UbcE2A with known ubiquitin-conjugating enzyme sequences revealed that ubcE2A is most homologous to Saccharomyces cerevisiae UBC9 (75% similarity) (45), Schizosaccharomyces pombe hus5 (82% similarity) (46), and the recently published human homologue of UBC9 (100% similarity) (47). S and M phase cyclins are degraded by UBC9, an essential nuclear ubiquitin-conjugating enzyme in budding yeast (45), and Schizosaccharomyces pombe do not grow when hus5 is mutated (46). Also, in yeast harboring the temperature-sensitive ubc9-1 mutation (45), ubcE2A rescued the ubc9-1 mutant from growth inhibition (data not shown). Because of the sequence homology and the functional complementation of the ubc9-1 mutant, we conclude that ubcE2A is the rat homologue of S. cerevisiae UBC9.
Cellular Localization of UbcE2A Protein-To localize the UbcE2A protein, we transfected monkey COS7 cells with a plasmid expressing HA-tagged UbcE2A and analyzed them by indirect immunofluorescence. A monoclonal anti-HA antibody (12CA5) and a rhodamine-tagged secondary antibody were used to detect HA-UbcE2A in transfected cells. The UbcE2A protein was expressed primarily in the nuclei (Fig. 1, left), as confirmed by counterstaining with Hoechst 33258. No staining was visible in vector-transfected cells (not shown). The immunoblot of nuclear extract from HA-UbcE2A-transfected cells (Fig. 1, right) showed a protein of 20 kDa, consistent with the expected molecular mass of UbcE2A. Because the UbcE2A protein localizes to the nucleus, it may act on E2A nuclear factors.
UbcE2A Binds to the E2A Proteins in Vitro-To confirm the interaction observed in yeast, we performed an in vitro binding assay. Radiolabeled, in vitro translated Id3 or UbcE2A was bound to GST-E12-(477-654) that had been immobilized on glutathione-Sepharose beads (Fig. 2). As anticipated, UbcE2A associated with GST-E12-(477-654) but not with GST. The interaction of Id3 with GST-E12-(477-654) served as a positive control. [ 35 S]Methionine-labeled, in vitro translated UbcE2A was also immunoprecipitated with an antibody to E12 in the presence of in vitro translated E12 protein (data not shown). We conclude that a specific interaction takes place between E12 and UbcE2A.
Interaction between UbcE2A and the E2A Proteins Is Specific-To study the specificity of the interaction between E12 and UbcE2A, we introduced full-length UbcE2A fused to the B42 transcription activation domain (AD-UbcE2A) into yeast cells containing various LexA fusion proteins. ␤-Galactosidase expression from the lacZ reporter gene increased by 20-fold in lysates from yeast bearing AD-UbcE2A and LexA-E12-(477-654) or LexA-E47-(477-651) ( Fig. 3; E12 and E47 versus Vector). This observation indicates that E12 and E47 interacted with UbcE2A equally well and that the primary amino acid sequence within the differentially spliced region was not crucial for binding. We examined the specificity of the interaction partners further by transforming yeast harboring expression plasmids encoding LexA fused to known HLH proteins. No interaction was detected with LexA fused to Id3 (33), the leucine zipper protein Max (34), or the homeodomain protein OCT-1 (35) (Fig. 3). Weak promoter activity was detected after introduction of LexA-c-Myc; however, LexA-c-Myc has been shown to cause higher background LacZ expression when studied with other proteins (48). In addition, we have observed no interaction between E12-(477-654) and UBCH5 (49), the human ubiquitin-conjugating enzyme involved in the ubiquitination of p53 (data not shown).
Map of UbcE2A-Interacting Regions-To map the E2A protein domain that binds to UbcE2A, we generated deletion mutants and assayed transcriptional activity by the yeast interaction trap. As anticipated, deletion of the basic or the HLH region had no effect on UbcE2A binding to E47 (Fig. 4, left). More extensive mapping localized the binding site to a 54amino acid region, E47-(477-530), 5Ј-proximal to the basic HLH domain (Fig. 4, left). This region is conserved in both E12 and E47. By itself this region conferred specific binding to UbcE2A; moreover, a construct lacking the E47-(477-538) region bound to Id3 but had no affinity for UbcE2A (Fig. 4, right; E47⌬-(477-538)). In contrast with this small interaction domain on E12/E47, almost the entire UbcE2A protein, including the conserved catalytic site, was required for binding to E12; only about 29 amino acids at the C terminus were dispensable (Fig. 5, left and right).
The E12 Protein Is Unstable-The specific binding of E12 by a ubiquitin-conjugating enzyme suggested that the E2A protein half-life may be regulated by proteolysis. We first studied E12 turnover to test the possibility that the protein was metabolically labile. COS7 cells transfected with a human E12 expression plasmid were pulse-labeled with [ 35 S]methionine for 60 min and then chased with unlabeled methionine for up to 120 min. E12 was immunoprecipitated from the lysates with an antibody to human E12 and analyzed by SDS-PAGE (Fig. 6A). Immunoprecipitation of the lysate revealed an M r 72,000 band migrating at the same position as E12 protein translated in vitro (Fig. 6A, E12IVT). These experiments showed that E12 is labile in vivo and has a half-life of about 60 min (Fig. 6B). Similar results were obtained with NIH3T3 cells (data not shown). Thus, E12 appears to be the target of an intracellular degradation pathway.
E12 Is Degraded through the Ubiquitin-Proteasome Pathway-We used the method described by Palombella et al. (26) without modification to test whether E12 is degraded by a proteasome. Forty-eight hours after COS7 cells had been transfected with a human E12 expression plasmid, they were treated for 1 h with the proteasome inhibitor MG132 or the protease inhibitor leupeptin (a negative control). MG132 stabilized the E12 protein, whereas leupeptin had no effect (Fig. 7). We conclude that degradation of E12 involves a proteasome.
Because protein degradation through a proteasome requires tagging of the protein by covalent attachment of multiple ubiquitin molecules (50), we next investigated whether E12 could be ubiquitinated in vivo by a method used to show ubiquitination of c-Jun (22). In these assays (Fig. 8), the E12 expression plasmid (pCR 3 E12) together with an HA-tagged ubiquitin expression plasmid (HA-Ub) were introduced into COS7 cells by transient transfection. c-Jun (pCR 3 jun), which is known to be multi-ubiquitinated (22), was used as a control. Equivalent amounts of lysate were immunoprecipitated with an antibody to E12 or c-Jun. The precipitated proteins were separated by SDS-PAGE, blotted onto Immobilon filters, and probed with a monoclonal antibody to HA (12CA5). The faint ladder of bands visible for c-Jun-transfected lysates above M r 39,000 (relative molecular mass of c-Jun) indicated formation of multiple ubiquitin conjugates (Fig. 8). A more distinct ladder of bands was visible for E12-transfected lysates. In both, most of the reactivity appeared above M r 200,000, which indicates significant multi-ubiquitination. Because E12 is ubiquitinated in vivo and proteasome inhibitors block its degradation, the ubiquitin-proteasome pathway appears to regulate the abundance of this transcription factor.
Overexpression of Antisense UbcE2A mRNA Stabilizes E12-To demonstrate the role of UbcE2A in E12 degradation more directly, we tested E12 expression by stably transfecting NIH3T3 cells with antisense ubcE2A cDNA (two antisense clones were studied, Asc3 and Asc6). Levels of 1.1-kilobase ubcE2A mRNA decreased in Asc3-and Asc6-transfected cells, to about 30 and 32%, respectively, the level in vector-transfected (control) cells, as measured by Northern blotting with a 32 P-labeled ubcE2A antisense riboprobe (data not shown). Pulse-chase analysis was performed 48 h after these cells had been transiently transfected with an E12 expression plasmid. In both antisense clones, the E12 protein was stabilized by approximately 2-fold in comparison with the vector clone ( Fig.  9). We conclude that UbcE2A plays an important role in regulating the level of E12 protein in cells.

DISCUSSION
UbcE2A Is a Novel Binding Partner of the E2A Proteins-UbcE2A, the ubiquitin-conjugating enzyme we isolated by the yeast two-hybrid system, interacts specifically with the HLH E2A proteins E12 and E47. Because of sequence homology and functional complementation, rat UbcE2A appears to be a homologue of yeast UBC9. Although UbcE2A does not contain an HLH domain, it binds E12 and E47 specifically (Figs. 3 and 4). Most of the UbcE2A molecule is necessary for binding to the E2A proteins; however, a 54-amino acid region in E47 (amino acids 477-530) located 5Ј of the basic and HLH domains is sufficient to bind UbcE2A. Thus we have identified a novel E2A interaction domain, amino acids 477-530, that binds to UbcE2A and may regulate E12/E47 turnover.
The E2A Proteins Are Highly Unstable-The specific binding of E12 by a ubiquitin-conjugating enzyme suggests that proteolysis may regulate the half-life of the E2A proteins. Two observations suggest that the E2A proteins may have a short half-life. First, increases in the amount of E12 or E47 in the mid-G 1 phase prevent entry into the S phase in serum-stimulated fibroblasts (13). Because the E2A proteins must be downregulated if the cell cycle is to progress, they must have a short half-life. Second, a feature of rapidly degraded proteins is the presence of PEST sequences, polypeptide chains rich in proline, glutamate/aspartate, serine, and threonine (51). Using the PEST-FIND program (14), we identified three PEST sequences in E12 (amino acids 47-67, 169 -189, and 521-537). Indeed, we found that E12 protein expression declined within 3 h of serum stimulation and became undetectable after 9 h (data not shown). By pulse-chase analysis, we found that E12 has a half-life of 60 min (Fig. 6B).
The E2A Proteins Are Degraded by the Ubiquitin-Proteasome Pathway through UbcE2A-Our observations indicate that the instability of the E2A proteins is mediated by the ubiquitinproteasome pathway. The 20 S proteasome inhibitor MG132 completely blocked degradation of E12 in experiments in which the protease inhibitor leupeptin was used as a control (Fig. 7), and E12 was multiply ubiquitinated in an in vivo assay (Fig. 8).
To our knowledge this is the first demonstration that the E2A proteins are degraded by the ubiquitin-proteasome pathway, which has also been shown to regulate other important transcription factors and cell cycle regulators (27). The specificity of substrate recognition by the ubiquitinproteasome pathway appears to be mediated by ubiquitin-conjugating enzymes, sometimes in conjunction with ubiquitin ligases. For example, a complex forms between the UBC6 and UBC7 enzymes in the ubiquitination pathway targeting degradation of the yeast transcription factor MAT␣2 (52). Only two genes encoding ubiquitin ligases have been cloned so far, S. cerevisiae UBRI and human E6-AP. The UBRI protein interacts with the RAD6 ubiquitin-conjugating enzyme to form a complex that targets substrates bearing "destabilizing" N-terminal residues (N-end rule substrates) (53). The E6-AP protein interacts with the E6 oncoprotein of human papilloma virus and induces ubiquitination and subsequent degradation of p53 (54). Although we detected a direct interaction between E12 and its ubiquitin-conjugating enzyme, we cannot completely rule out the possibility that E12 ubiquitination requires an unknown ubiquitin ligase.
We found that ubcE2A mRNA expression is regulated differentially in fibroblasts (data not shown). Expression is maximal at the mid-G 1 phase, before the onset of the S phase. Our finding is consistent with the observation that E12 is degraded during progression of the cell cycle (13). To maintain oscillating levels of regulatory proteins, the ubiquitin-conjugating machinery would have to be activated only at specific points in the cell cycle. For example, the yeast CDC34 ubiquitin-conjugating enzyme, which is required for the transition from the G 1 to the S phase, is regulated by phosphorylation and ubiquitination (55). Although it is conceivable that UbcE2A is subject to similar modification (UbcE2A contains four putative phosphoryla-tion sites), our observation that it is up-regulated during G 1 suggests that the abundance of UbcE2A, and hence ubiquitination, may determine the rate of E12 turnover.
Specific ubiquitin-conjugating enzymes are necessary for the degradation of many cellular substrates. p53 degradation requires the human homologue of UBC4 but not that of UBC2 (54), and p27 degradation specifically involves the human homologues of UBC2 and UBC3 (24). These observations suggest that it may be possible to inhibit degradation of a substrate in vivo by inhibiting its specific ubiquitin-conjugating enzyme. Indeed, microinjection of an antisense UBC4 expression plasmid into human tumor cells containing high levels of the p53 protein inhibited E6-stimulated degradation of p53 (54). We demonstrate here by an antisense approach that down-regulation of UbcE2A expression inhibits degradation of E12 (Fig. 9). Because the tissue-specific gene transcription that moves cells from a proliferative to a differentiated state involves the ubiquitous E2A proteins, it may be possible to regulate cellular differentiation by targeting UbcE2A. FIG. 9. Inhibition of E12 degradation in cells transfected with antisense UBCE2A. Cells were stably transfected with pCR 3 (Vector) or antisense ubcE2A expression plasmids (Asc3 and Asc6). The cells were then transiently transfected with a human E12 expression plasmid and analyzed by pulse-chase as described for Fig. 6. Results from a representative experiment are shown. The experiment was repeated twice with similar results.