(Received for publication, October 22, 1996)
From the Cardiovascular Biology Laboratory, 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.
By alternative splicing, the E2A gene encodes two
proteins, E12 and E47, through two adjacent exons encoding a basic
helix-loop-helix (HLH)1 motif (1). These
proteins belong to a family of eukaryotic transcription regulators
distinguished by the highly conserved HLH motif, which mediates
dimerization, and by the adjacent basic region, which mediates
site-specific DNA binding (2, 3). The ubiquitous E2A proteins form
heterodimers with tissue-specific HLH proteins that then bind to DNA
and up-regulate the transcription of target genes. Tissue-specific HLH
proteins include the MyoD family involved in skeletal muscle
differentiation (4), the achaete-scute family 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 G1-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 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
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.
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 pCR3 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
pCR3 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 MASYPYDVPDYASPEF 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.
All cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal calf serum
(Hyclone), 100 units/ml penicillin, and 100 mg/ml streptomycin in a
humidified atmosphere at 37 °C with 5% CO2. Mouse
monoclonal anti-HA antibody (12CA5) was purchased from Berkeley
Antibody Co. (Richmond, CA), anti-human E12/E47 monoclonal antibody
from Pharmingen (San Diego, CA), anti-human E12 rabbit polyclonal
antibody, and anti-mouse c-Jun antibody from Santa Cruz Biotechnology
(Santa Cruz, CA), goat anti-mouse IgG-HRP from Amersham Corp., and
rhodamine-conjugated anti-mouse IgG from Kirkegaard & Perry
Laboratories (Gaithersburg, MD). Normal rabbit and mouse sera were
purchased from ICN Biochemicals (Costa Mesa, CA).
We screened for
E12-interacting proteins by the yeast two-hybrid interaction trap
according to Gyuris et al. (37). EGY48 (MAT 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-
Crude extracts were assayed for yeast 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 µl 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.
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 µg/ml) and incubated (with shaking) for
3-5 h at 37 °C until the A600 reached 0.8. Isopropyl 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 µCi/ml
[35S]Met for 60 min at 37 °C. Cells were chased in
warm DMEM supplemented with 100 µg/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 µM) was added 1 h before pulse-chase and was present
throughout the pulse-chase 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 CompleteTM
protease inhibitor mixture (Boehringer Mannheim)) for 20 min at
4 °C. Lysates were cleared of nuclei and debris by centrifugation at
14,000 × 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. 35S 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 µg 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).
COS7 cells were electroporated
with 6 µg of the E12 or c-Jun expression construct and 20 µg of the
HA-tagged 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.).
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 × 106 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
ubiquitin-conjugating 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.
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.
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. [35S]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.
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. 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 54-amino acid region, E47-(477-530),
5
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
[35S]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 Mr 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.
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
(pCR3E12) together with an HA-tagged ubiquitin expression
plasmid (HA-Ub) were introduced into COS7 cells by transient
transfection. c-Jun (pCR3jun), 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 Mr 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
Mr 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.
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 32P-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.
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 5 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-G1 phase prevent entry
into the S phase in serum-stimulated fibroblasts (13). Because the E2A
proteins must be down-regulated 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).
Our observations indicate that the instability of
the E2A proteins is mediated by the ubiquitin-proteasome 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 ubiquitin-proteasome
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 We found that ubcE2A mRNA expression is regulated
differentially in fibroblasts (data not shown). Expression is maximal
at the mid-G1 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 G1 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 phosphorylation sites), our observation that it is
up-regulated during G1 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U54632[GenBank]. We thank Roger Brent and Russell Finley
(Massachusetts General Hospital, Boston) for plasmids, yeast strains,
and antibody for the interaction trap; Alfred Goldberg and Olivier Coux
(Harvard Medical School, Boston, MA) for proteasome inhibitors and
helpful suggestions; Stefan Jentsch and Petra Hubbe (Zentrum für
Molekulare Biologie, Heidelberg University, Germany), Fiorenzo Peverali
(European Molecular Biology Laboratory, Heidleberg, Germany), and
Mathias Treier (EMBL) for plasmids and yeast strains; David Baltimore (Massachusetts Institute of Technology, Cambridge) and Zhengsheng Ye
(The Rockefeller University, New York) for plasmids and technical assistance; and Martin Rechsteiner (University of Utah School of
Medicine, Salt Lake City) for the PEST-FIND program. We are also
grateful to Bonna Ith for cell culture and Thomas McVarish for
editorial assistance.
Cardiac Unit, Massachusetts General Hospital,
Boston, Massachusetts 02114
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
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 I
B
(25, 26).
-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).
Plasmids
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 (in-frame) 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 × 106 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 × 106 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.
-D-galactoside, and either
2% glucose or 2% galactose, and 1% raffinose. We checked the color
of the yeast 48 h later.
Fig. 3.
Specificity of UbcE2A interactions in yeast
by -galactosidase assay. Cells of the S. cerevisiae
strain EGY48/pSH18-34 were sequentially transformed with the indicated
LexA fusion plasmid (Baits) and the activation domain (AD-UbcE2A)
library isolate. At least three independent colonies from each
AD-UbcE2A/LexA fusion protein pair were used to inoculate a
galactose-containing liquid culture.
-Galactosidase activity
expressed from the lacZ reporter gene (normalized units) was
measured; error bars indicate standard deviations.
Expression of the appropriate LexA fusion proteins was confirmed by
Western blotting (data not shown).
[View Larger Version of this Image (14K GIF file)]
-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(A420)/(time(min)·vol(ml)·A600).
-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 × g for 5 min at 4 °C.
Aliquots (1 ml) of bacterial supernatant were rocked for 30 min at
4 °C with 25 µl of glutathione-Sepharose 4B (Pharmacia). The
Sepharose beads were then washed three times with PBS.
35S-Labeled proteins were generated by using the TNT
T7-coupled reticulocyte lysate system (Promega) with the Id3 or UbcE2A
expression construct in pCite4 (Novagen). 35S-Labeled
protein (3 µl) was incubated with the beads (25 µl) 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.
A Ubiquitin-conjugating Enzyme Cloned by the Yeast Interaction
Trap
Fig. 1.
Nuclear localization and functional activity
of UbcE2A. COS7 cells transfected with hemagglutinin (HA)-tagged
UbcE2A (HA-UbcE2A) were fixed and stained with anti-HA antibody 12CA5 and rhodamine-conjugated secondary antibody. Staining with Hoechst 33258 shows the position of the nuclei. The immunoblot on the right
shows nuclear extracts prepared from HA-UbcE2A-transfected or
mock-transfected cells stained with 12CA5. Control was total extract
prepared from yeast expressing HA-UbcE2A. The arrow
indicates HA-UbcE2A protein.
[View Larger Version of this Image (52K GIF file)]
Fig. 2.
Specificity of UbcE2A interactions in
vitro by GST assay. Bacterially produced GST-E12-(477-654)
or GST alone was bound to glutathione-Sepharose beads and incubated
with [35S]methionine-labeled Id3 or UbcE2A produced by
in vitro translation. Specifically bound protein was
resolved by SDS-PAGE. There was at least 10 times more GST than
GST-E12-(477-654) on a gel stained with Coomassie Blue (data not
shown).
[View Larger Version of this Image (45K GIF file)]
-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).
-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).
Fig. 4.
Binding of LexA-E47 fusion baits to
full-length AD-UbcE2A interactant protein. Left, E47 regions
used as baits in yeast interaction trap assay. The basic domain is
black; the HLH domain is striped. The
asterisk above the Ala mutant marks the five amino acid
substitutions in the basic domain. In each case, at least six
independent transformants were scored for the intensity of galactose-inducible blue that developed in the presence of
5-bromo-4-chloro-3-indolyl--D-galactoside: +++,
dark blue; +/
, faint blue flecks in some colonies;
,
white colonies only. Right,
-galactosidase activity in
lysates prepared from transformants harboring the indicated protein
pairs.
-Galactosidase levels were measured in duplicate from three
independent isolates; average value is shown.
[View Larger Version of this Image (18K GIF file)]
Fig. 5.
Binding of UbcE2A fusion proteins to the
E12-(477-654) fusion protein bait in yeast interaction trap
assay. Left, UbcE2A regions used as interactants.
Striped box indicates conserved catalytic domain of
ubiquitin-conjugating enzymes. Right, -galactosidase levels in yeast expressing the indicated protein pairs.
-Galactosidase levels were measured in duplicate from three
independent isolates; average value is shown.
[View Larger Version of this Image (15K GIF file)]
Fig. 6.
Pulse-chase analysis of E12 in transfected
COS7 cells. A, cells expressing human E12 were labeled with
[35S]methionine for 1 h and then chased with
unlabeled methionine for the indicated times. Clarified cell lysates
(3 × 105 cpm each) were subjected to
immunoprecipitation with an anti-E12 antibody and analyzed by SDS-PAGE
fluorography. [35S]Methionine-labeled, in
vitro translated E12 (E12IVT) marks the position of the E12
protein. No signal was obtained from vector-transfected cells
immunoprecipitated with anti-E12 antibody (Vector) or E12-transfected cells immunoprecipitated with preimmune serum (Preimmune).
The fluorogram is from a typical experiment. B, profile of
the E12 half-life obtained by PhosphorImaging analysis of the bands in A.
[View Larger Version of this Image (13K GIF file)]
Fig. 7.
The proteasome inhibitor MG132 blocks
degradation of E12 in vivo. Monkey COS7 cells were
electroporated with a human E12 expression plasmid. After 48 h,
cells were treated with the proteasome inhibitor MG132,
dimethyl sulfoxide (DMSO) (an MG132 diluent), or leupeptin
for 1 h. They were then pulse-chased for 3 h with
[35S]methionine. Cell extracts were immunoprecipitated
with an anti-E12 antibody and analyzed by SDS-PAGE fluorography.
Inhibitors were present throughout the pulse-chase period. The signal
intensity of the E12 bands was measured by PhosphorImaging.
[View Larger Version of this Image (13K GIF file)]
Fig. 8.
Ubiquitination of nontagged c-Jun and
E12. COS7 cells were electroporated with cytomegalovirus vectors
(pCR3) directing synthesis of HA-tagged
ubiquitin (HA-Ub) and nontagged c-Jun
(pCR3jun) or E12
(pCR3E12). Cell extracts were
immunoprecipitated with an anti-E12 antibody, eluted, separated by
SDS-PAGE, and immunoblotted with a mouse monoclonal antibody to HA
(12CA5). The arrow marks mouse immunoglobulin heavy chain
that cross-reacted with the secondary antibody. Brackets to
the right of the panels mark a smear of cross-reactive
material corresponding to the size of ubiquitinated c-Jun and E12. The
bottom right panel shows E12 expression in transfected
cells.
[View Larger Version of this Image (31K GIF file)]
Fig. 9.
Inhibition of E12 degradation in cells
transfected with antisense UBCE2A. Cells were stably
transfected with pCR3 (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.
[View Larger Version of this Image (55K GIF file)]
UbcE2A Is a Novel Binding Partner of the E2A Proteins
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.
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.
*
This work was supported in part by a grant from
Bristol-Myers Squibb. The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by National Institutes of Health Grant R01GM
53249.
**
To whom all correspondence should be addressed: Cardiovascular
Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-1010; Fax: 617-432-4098; E-mail:
haber{at}cvlab.harvard.edu.
1
The abbreviations used are: HLH,
helix-loop-helix; UbcE2A, ubiquitin-conjugating enzyme that binds the
E2A proteins; AD-UbcE2A, full-length UbcE2A fused to the B42
transcription activation domain; HA, hemagglutinin; HA-Ub, HA-tagged
ubiquitin expression plasmid; GST, glutathione
S-transferase; DMEM, Dulbecco's modified Eagle's medium;
E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3,
ubiquitin-protein ligase; E12IVT, E12 protein translated in
vitro; Asc3 and Asc6, antisense ubcE2A clones; PCR,
polymerase chain reaction; PAGE, polyacrylamide gel
electrophoresis.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.