(Received for publication, June 7, 1995; and in revised form, September 19, 1995)
From the
Two very closely related human E2 ubiquitin conjugating enzymes, UbcH5B and UbcH5C, have been identified. These enzymes are products of distinct genes and are 88-89% identical in amino acid sequence to the recently described human E2, UbcH5 (now designated UbcH5A). UbcH5A-C are homologous to a family of five ubiquitin conjugating enzymes from Arabidopsis thaliana, AtUBC8-12. They are also closely related to Saccharomyces cerevisiae ScUBC4 and ScUBC5, which are involved in the stress response, and play a central role in the targeting of short-lived regulatory proteins for degradation. mRNAs encoding UbcH5A-C were co-expressed in all cell lines and tissues evaluated, with UbcH5C transcripts generally expressed at the highest levels. Analysis of Southern blots suggests that there are likely to be other related members of this family. Both UbcH5B and UbcH5C form thiol ester adducts with ubiquitin, and have activities similar to UbcH5A and AtUBC8 in the conjugation of ubiquitin to target proteins in the presence of the human ubiquitin protein ligase E6-AP. These results establish the existence of a highly conserved, and widely expressed, family of human ubiquitin conjugating enzymes.
The modification of proteins with ubiquitin constitutes an
important cellular mechanism for targeting proteins for degradation by
the 26 S proteasome (reviewed in (1) ). Proteins known to be
degraded in this fashion include abnormal polypeptides and a number of
short-lived regulatory proteins including plant phytochrome
A(2) , c-Myc(3) , c-Jun(4) ,
cyclins(5) , p53(6) , and components of the NF-B
complex(7) . In Saccharomyces cerevisiae, this
modification has been extensively studied and found to lead to the
degradation of abnormal and test proteins, with the susceptibility of
some proteins to ubiquitin-mediated degradation dependent on the nature
of their amino termini (reviewed in (8) ). Several
transmembrane receptors are also ubiquitinated specifically in response
to receptor
engagement(9, 10, 11, 12) , but in
these cases, the relationship between ubiquitination and degradation is
less clear. Ubiquitinated proteins are found at increased levels in
neuropathological states including Alzheimer's
disease(13) .
At least three classes of enzymes are involved in the conjugation of ubiquitin to proteins. Ubiquitin activating enzyme (E1), is responsible for the ATP-dependent charging of ubiquitin through the formation of a high energy thiol ester bond between the carboxyl terminus of ubiquitin and a cysteine within E1. The thiol ester-linked ubiquitin is transferred from E1 to a cysteine residue in an E2, or ubiquitin conjugating enzyme. E2 enzymes either by themselves or in combination with E3 enzymes (ubiquitin protein ligases) then transfer ubiquitin monomers or multiubiquitin chains to target proteins, where stable isopeptide linkages are formed (reviewed in Refs. 1 and 14-17).
E1 enzymes have been characterized in several species including yeast (18) , wheat(19) , and man(20) . Thus far, two gene products encoding E3 enzymes have been cloned, one is from S. cerevisiae(21) , and the other is a human gene product termed E6-AP, named because it associates with the human papilloma virus E6 oncoprotein(22) . This E3 has been shown to catalyze the E6-dependent ubiquitination of p53(6) . E3 activities have also been characterized in rabbit reticulocytes (23) and in wheat(24) .
A multitude of E2s exist. In S. cerevisiae there are at least 10 E2 genes(16) , whereas in Arabidopsis thaliana over 30 are likely present(17) . Functions for these enzymes include roles in DNA repair, cell cycle progression, organelle biogenesis, secretion, and stress response (reviewed in (16) ). There is at least one example where two ubiquitin conjugating enzymes function in concert to transfer ubiquitin to a specific target protein(25) . Two closely related S. cerevisiae E2s, ScUBC4 and ScUBC5, play important roles in the turnover of normal and abnormal proteins(16, 26) . The levels of ScUBC4/5 are increased in response to stress, and their loss has severe effects on cellular functions; the concomitant loss of a third E2, ScUBC1, is lethal. A single homolog of ScUBC4/5 has been characterized in Caenorhabditis elegans(27) and in Drosophila melanogaster(28) . In A. thaliana, a set of five closely related gene products with over 88% similarity to ScUBC4/5 and over 94% similarity to each other have been identified, with genomic evidence for at least one additional family member (29) .
More recently a human gene product that is 78-79% identical to ScUBC4/5 in amino acid sequence has been identified. This enzyme, UbcH5 (now designated UbcH5A), stimulates the conjugation of ubiquitin to the tumor suppressor p53 in the presence of E6-AP and E6(30) . At least one member of the A. thaliana family of ScUBC4/5-related ubiquitin conjugating enzymes, AtUBC8, also can serve in this role, while an unrelated A. thaliana E2 does not(30) . In this study we report the characterization of two additional human members of this class of closely related and highly conserved ubiquitin conjugating enzymes.
Genomic DNA from human placenta
(Oncor, Gaithersburg, MD) and from Balb/c mice was exhaustively
digested prior to separation on 0.8% agarose gels. Blots were
transferred to nylon-backed nitrocellulose (Schleicher and Schuell) and
hybridized as above with PCR-generated probes (Bios, New Haven CT)
prior to a final wash at 0.2 SSC at 63 °C.
Reaction mixtures
containing the various E2s (0.05 to 10 µl of bacterial lysate), 0.1
µg of purified recombinant wheat E1, 0.5 units of phosphocreatine
kinase, and 0.9 µg of I-ubiquitin were incubated with
0.1 µl of either E6-AP extract, extract from mock infected cells,
or H
O in 20 µl of 50 mM Tris-HCl (pH 7.5), 0.2
mM ATP, 0.5 mM MgCl
, 0.1 mM dithioerythritol, 1 mM creatine phosphate for the
indicated times at 30 °C. Reactions were terminated by boiling
samples for 10 min in SDS-PAGE sample buffer containing 4%
-mercaptoethanol. Samples were subjected to SDS-PAGE and gels were
stained by Coomassie Brilliant Blue, dried between cellophane, and
visualized by autoradiography.
Prior to the initiation of this study, no mammalian homologs
of the yeast ScUBC4/5 and the A. thaliana AtUBC8-12 E2
enzymes had been identified. To determine if there are related members
of this E2 class in man, oligonucleotides based on conserved regions in
ScUBC5 and the related D. melanogaster DmUbcD1 were
synthesized. Similar oligonucleotides had previously been used for the
identification of a C. elegans E2, CeUbc-2, by
PCR(27) . The use of these oligonucleotides in the PCR with
first strand cDNA from PBL as template resulted in a 325-base pair
fragment. Cloning and sequencing of this fragment revealed a product
with an open reading frame that was most homologous to the region
encoding amino acids 52 to 139 of DmUbcD1 and CeUbc-2. Using DNA
prepared from a human PBL library as template, PCR was carried out in
which sense and antisense oligonucleotides synthesized based on the
sequence of the 325-base pair fragment were paired with
oligonucleotides corresponding to the T3 or T7 regions flanking the
Zap polylinker. Overlapping fragments extending 5` and 3` were
identified, sequenced, and found to contain parts of the original
product. Oligonucleotides based on regions predicted to be the 5`- and
3`-untranslated regions were then synthesized and used to clone the
entire open reading frame by PCR (Fig. 1). A search of the
GenBank data base found that the deduced amino acid sequence of this
gene product was 89% identical to a recently described human E2, UbcH5 (30) (now designated UbcH5A), and was thus designated UbcH5B.
Figure 1: Sequence alignment of UbcH5B and UbcH5C. Sites of translation initiation and stop codons are underlined.
Oligonucleotides generated based on the original 325-base pair PCR fragment also yielded overlapping fragments that were distinct from UbcH5B. This second sequence was truncated at its 5` end prior to the predicted site of translation initiation, thus it did not encode a complete E2 enzyme. However, it appeared to overlap a partial sequence found in GenBank that had been designated EST06924. EST06924 had previously been identified by random sequencing of a human infant brain cDNA library (accession number: T09032)(37) , and had been noted to have homology to the 5` ends of cDNAs encoding ubiquitin conjugating enzymes. A 5`-oligonucleotide based on EST06924 was synthesized and used in combination with an antisense oligonucleotide based on the 3` region of our sequence in the PCR, with DNA from the PBL library as template. This resulted in the generation of a full-length clone encoding an E2 related to UbcH5A and UbcH5B that we denote UbcH5C. The correct sequence of this clone was independently confirmed by PCR amplification and cloning from a cDNA library made from YT-1, a human natural killer cell line.
The cDNAs encoding UbcH5B and UbcH5C are 87% identical on a nucleotide level within the predicted coding region. In contrast, their 3`-untranslated regions are only 23% conserved. Both UbcH5B and UbcH5C are 78% identical to UbcH5A within their coding regions. Alignment of the deduced amino acid sequences of UbcH5B and UbcH5C shows only four amino acid differences, the only non-conservative change being amino acid 11 where there is an asparagine in UbcH5B and a serine in UbcH5C. When compared to the amino acid sequence of UbcH5A, UbcH5B is 89%, and UbcH5C is 88% identical (Fig. 2). These three human E2s are 77-80% identical to ScUBC4/5 (26) and to members of the AtUBC8-12 family of E2s(29) . Although the homology between UbcH5A and the two new family members is substantial, the amino acid sequences of UbcH5B and UbcH5C are even more closely related to the D. melanogaster DmUbcD1(28) , and C. elegans CeUbc-2(27) , where there is 92-95% identity. To determine whether UbcH5A-C are the results of polymorphism among humans, 5`- and 3`-oligonucleotides based on the published UbcH5A sequence were generated and used to isolate cDNAs encoding UbcH5A from both the PBL and YT libraries. The PBL isolate was sequenced and found to be identical to the published sequence(30) .
Figure 2: Amino acid comparison of closely related ubiquitin conjugating enzymes. Deduced amino acid sequence of UbcH5B and UbcH5C and comparison to UbcH5(A), and closely related E2s from D. melanogaster (DmUbcD1), C. elegans (CeUbc-2), S. cerevisiae (ScUBC4/5), and A. thaliana (AtUBC8/9). Amino acids that are identical among all members of the family are shaded. The percent identity of UbcH5A-C to the related E2s is shown below the alignment.
To determine
the tissue distribution and relative expression of transcripts encoding
UbcH5A-C, first strand cDNA was synthesized from RNA followed by PCR
amplification with pairs of oligonucleotides specific to individual
members of the family (Fig. 3A). The resultant products
were hybridized with a P-labeled 60-base oligonucleotide
probe that was equally mismatched against UbcH5A-C (Fig. 3A), and therefore should hybridize equally well
with PCR products encoding UbcH5A-C. To establish the specificity of
the oligonucleotide pairs and the ability of the probe to hybridize
with the PCR products, amplification of linearized plasmid DNA encoding
each of the three E2s was carried out. As shown (Fig. 3B), the oligonucleotide pairs behaved as
expected, with products detected only in samples having the appropriate
combination of oligonucleotide and template. To assess the expression
of UbcH5A-C in samples reverse-transcribed from human RNA, conditions
were optimized to achieve a degree of amplification that was within a
linear range. A typical result using 25 cycles of PCR and three
different dilutions of template is shown (Fig. 3C). Results
obtained using 1:16 dilutions of first strand cDNA are presented in Fig. 3D. For each source of RNA, the values obtained
for UbcH5B and UbcH5C were normalized to UbcH5A. The results of this
analysis demonstrate that messages encoding UbcH5A-C are co-expressed
in a number of different tissue and cell lines (Fig. 3D). While UbcH5C was consistently found at
higher levels than UbcH5A, the ratio of these two family members varied
from 9:1 (PBL) down to 2:1 (prostate and HeLa). In most cases UbcH5B
was found at levels between UbcH5A and UbcH5C. However, there was
significant variation in the expression of UbcH5B relative to UbcH5A
and UbcH5C. For example, in PBL, the level of UbcH5B was almost equal
to UbcH5C, while in other samples, such as Jurkat and prostate, UbcH5B
more closely approximates UbcH5A.
Figure 3:
Expression of mRNA for UbcH5A-C in human
cell lines. A, schematic representation of oligonucleotides
used for selective amplification of UbcH5A-C. Oligo pairs are
designated A, B, or C, based on their predicted ability to amplify
UbcH5A-C. The position of the 60-base probe used for hybridization is
indicated by the hatched area (see ``Materials and
Methods'' for specific sequences). B, specificity of
amplification. Plasmid DNA encoding UbcH5A-C was linearized and 0.2 pg
subject to PCR amplification for 25 cycles with each of the three oligo
pairs. Amplified fragments were resolved on 1.5% agarose gels followed
by transfer to nitrocellulose membrane and hybridization with an
end-labeled 60-base probe that was equally mismatched against the three
members of the family (see ``Materials and Methods'' for
details regarding hybridization and wash conditions). C, total
RNA from colon DNA was reverse transcribed and various dilutions of
first strand cDNA subject to 25 cycles of PCR amplification with each
set of oligonucleotide pairs as described under ``Materials and
Methods.'' This was followed by hybridization with the
oligonucleotide probe described above. D, quantification of
the relative expression of UbcH5A-C. PCR products generated as
described for C were quantified by PhosphorImager. After
subtraction of background the values obtained for UbcH5B and UbcH5C for
each tissue or cell type were normalized to UbcH5A (represented by the thick line). The data shown are representative of at least two
independent PCR reactions carried out at a dilution of 1:16 of first
strand cDNA. In all cases multiple dilutions of first strand cDNA were
amplified to assure that the results used for this analysis were within
a linear range. &cjs2112;, UbcH5B; ,
UbcH5C.
To determine if there are other closely related members of the UbcH5A-C gene family, Southern blots were carried out on genomic DNA from human placenta and from Balb/c mice. DNA was digested with either HindIII or EcoRI and the resultant blots hybridized first with a radiolabeled probe corresponding to nucleotides 12 to 213 within the coding region of UbcH5C, and then re-hybridized with a probe corresponding to nucleotides 203 to 442 of UbcH5C (Fig. 4). Since the two probes have only an 11-base overlap, fragments that hybridized with both probes should give the minimal number of genes in this family. Up to five common bands were found in man and nine in mouse. The presence of fewer bands in the human HindIII lane than in the EcoRI lane may indicate that members of this family are closely linked in the human genome, and contained within a single fragment. Alternatively, preserved HindIII sites may exist between two genes in this family, resulting in co-migrating fragments. While some of the larger hybridizing bands present in murine DNA may represent distinct genes, given their relative intensity, they may also represent partially digested DNA. Even if these higher molecular weight murine species are discounted, it would appear that mammalian genomes encode more than three members of this E2 family.
Figure 4:
Southern analysis of UbcH5 genes. Genomic
DNA from human placenta or from inbred Balb/c mice was digested with
either HindIII (H) or EcoRI (E),
subjected to agarose gel electrophoresis, transferred to
nitrocellulose, and hybridized with a probe corresponding to bases 12
through 213 of UbcH5C and washed at 0.2 SSC at 63 °C. Blots
were stripped of radioactive material and re-hybridized with a second
probe corresponding to bases 203 to 442 of UbcH5C and washed under
similar conditions. Shown are the results of the first hybridization,
with an asterisk (*) indicating fragments that hybridized with
both probes.
To evaluate the
ability of these enzymes to function in ubiquitin conjugation, UbcH5A-C
were expressed in E. coli using the pET expression
system(38) . The expression of these gene products in E.
coli is shown in Fig. 5A. While all three cDNAs
resulted in species of the predicted size (16 kDa), lower levels
of UbcH5A were consistently found. A more slowly migrating major
species (
28 kDa) was also seen in lysates of E. coli expressing UbcH5A. A similar species of unknown significance was
found in the original description of this enzyme(30) . An
essential characteristic of E2s is their ability to form thiol ester
adducts with ubiquitin in an E1-dependent manner. All three of these
proteins were able to form thiol ester adducts with ubiquitin in the
presence of ATP and E1 (Fig. 5B). As expected for a
thiol ester linkage, the adducts were unstable in the presence of the
reducing agent
-mercaptoethanol.
Figure 5:
Expression and thiol ester formation of
UbcH5A-C. Panel A is a Coomassie Blue stain of crude extracts
of E. coli expressing UbcH5A-C after induction with
isopropylthio--galactosidase, compared to E. coli transformed with empty pET-15b vector. The 16-kDa band in the UbcH5A-C lanes represents recombinant E2. The prominent 28-kDa
band in the UbcH5A lane is of unknown significance. Panel B is
an anti-ubiquitin immunoblot showing thiol ester formation of UbcH5
family members with ubiquitin. Conjugate assay was carried out in the
presence or absence of E1 from wheat(19) . Samples were heated
to 95 °C in the presence or absence of
-mercaptoethanol prior
to resolution on SDS-PAGE, and immunoblotting as
described(35) .
The data presented in Fig. 5demonstrates that these enzymes are able to form thiol ester linkages with ubiquitin. To determine whether they are able to function with an E3 enzyme in the ubiquitination of target proteins, UbcH5A-C were tested for their ability to conjugate ubiquitin to cellular proteins in an E6-AP dependent manner. They were compared to AtUBC8, an E2 previously demonstrated to function with E6-AP in a cell-free system(6, 30) . Using a quantitative thiol ester assay, the volume of E. coli extract that had an equal activity for each E2 was determined (Fig. 6A). When equivalent amounts of thiol ester forming activity was added to conjugation assays, all four E2s demonstrated similar abilities to catalyze the transfer of ubiquitin to higher molecular weight proteins in an E6-AP-dependent fashion (Fig. 6B). Over the time period in which conjugate formation was linear (Fig. 6C), there was no substantial difference in the ability of these enzymes to catalyze the ubiquitination of cellular substrates.
Figure 6:
Ubiquitin conjugation of UbcH5A-C. To
carry out conjugation assays, volumes of lysate from E. coli expressing the various E2s being assayed were determined that
resulted in equivalent thiol ester adduct formation in the presence of
E1. Reaction volumes were equalized using lysate from E. coli not expressing an E2. Panel A demonstrates equivalent
thiol ester adduct formation between I-ubiquitin and the
indicated E2s. Adducts of
I-ubiquitin with E2s and with
E1 are indicated. Panel B illustrates the conjugation of
cellular proteins with
I-ubiquitin in an E6-AP and
E2-dependent fashion using equivalent thiol ester adduct activity for
the four different E2s. After incubation as indicated for 2 h at 30
°C, samples were heated to 95 °C in sample buffer containing
-mercaptoethanol, and resolved by SDS-PAGE. E. coli transformed with empty pET-15b vector is shown as a control.
Assays were carried out with lysates from Trich Ni cells that express
E6-AP. Lysates from cells not expressing E6-AP are shown as controls (Mock). As an additional control, samples to which no E.
coli lysate was added are shown (No E2 lanes). The
species seen in the AtUBC8 lanes at
25 and 32 kDa were found
reproducibly, and are of unknown significance. In Panel C,
conjugate formation was carried out for the indicated times. The area
of the gel corresponding to molecular masses greater than 50 kDa was
excised and quantified by
-counting.
, UbcH5B;
,
UbcH5C;
, UbcH5A;
, AtUBC8.
An increasing number of cellular proteins are being
recognized as substrates for conjugation to ubiquitin (reviewed in (1) ). In some cases, the nature of the amino terminus
determines the potential susceptibility for ubiquitin-mediated
degradation, a concept known as the N-end rule(8) . For the
mitotic cyclins and c-Jun, specific internal sequences have been
identified that confer susceptibility to
ubiquitination(4, 5) . However, in most cases, the
molecular mechanisms responsible for targeting proteins for
ubiquitination are unknown. Specificity with regard to the recognition
of targets would seem to lie with the E2 and E3 enzymes. In yeast and
in plants, a multitude of E2s exist(16, 17) . In
humans, several distinct E2s have been characterized. Two very closely
human related E2s, HHR6A and HHR6B, have been identified that are
homologous to the product of the yeast DNA repair gene RAD6(39, 40, 41) . In addition to
its function in DNA repair(42) , the RAD6 gene product
is also believed to be involved in the recognition of substrates via
the N-end rule(43, 44) . Another human E2, identified
by complementation studies, is the human homolog of the S.
cerevisiae CDC34, which is important in the G to S
transition in yeast(45, 46) . UbcH2, a human homologue
of AtUBC4-6 (47) and ScUBC8(48) , may be involved
in the ubiquitination of histones in an E3-independent manner (49) . These E2s are characterized by acidic COOH termini that
help in the recognition of basic histones. Studies of auto-antibodies
expressed in pemphigus foliaceus has resulted in the identification of
a human cDNA denoted EPF-5, which, in an alternative reading frame
(EPF5-ORF2), encodes an E2 enzyme(50) . The significance of
this reading frame remains to be determined.
It is clear from the present study that there are at least three related human E2s that are homologous to yeast ScUBC4/5 and A. thaliana AtUBC8-12. All three of these enzymes are expressed in the same cells, and all function in an equivalent manner in a cell-free system with the human E3 enzyme E6-AP. These findings naturally raise the issue of whether these enzymes perform redundant, partially overlapping, or distinct in vivo functions. Pertinent to a discussion of this issue is one of two related studies published while this manuscript was being prepared. In this study, Wing and Jain (51) describe two rat E2s, RnE2/2E and RnE2/4A, cloned from testis, that are identical in amino acid sequence to UbcH5B and UbcH5C, respectively. There is also greater than 95% nucleic acid conservation between each pair of human and rat cDNAs, this includes both the coding regions and the 3`-untranslated regions. A dendrogram, based on deduced amino acid sequence, illustrating the relationship between UbcH5A-C, related E2s from other species, and other human E2s is depicted in Fig. 7. It shows the close relationship that these ScUBC4/5-related E2s have to each other, and also suggests that genes encoding UbcH5B/C and UbcH5A likely arose by duplication quite early in evolution. The total conservation of amino acid sequence between UbcH5B/C and their rat equivalents, the early divergence of UbcH5B/C from the closely related UbcH5A, together with the finding that the relative levels of transcripts encoding UbcH5A-C varies among tissues and cell types makes it reasonable to surmise that there are likely to be in vivo differences in functions among these enzymes.
Figure 7: Evolutionary relationship of ubiquitin conjugating enzymes. This illustrates the likely evolutionary relationship between human E2s, designated by an asterisk (*), and E2s related to UbcH5A-C from other species (only two of the five A. thaliana members of this family are shown). Rn refers to Rattus norvegicus. This analysis was generated using GeneWorks (Intelligenetics, Mountainview, CA), using a progressive alignment method.
Based on genomic
analysis, and the precedent in A. thaliana, there are probably
other related members of this family of E2 enzymes. A second, recently
published relevant study reports the cloning of a human cDNA from the a
human cervical carcinoma cell line, HeLa. This cDNA encodes a protein
identical to UbcH5B except for the substitution of a glutamine for a
lysine at position 128 due to a single base substitution(52) .
This lysine is conserved in all of the ScUBC4/5-related E2s (Fig. 2) and was found in four distinct cDNAs encoding UbcH5B,
isolated from three sources, including two normal individuals. There
are only two additional base changes in the coding regions between the
HeLa-derived cDNA and UbcH5B, both of which are silent. Thus, while it
is conceivable that this (128KQ) form of UbcH5B represents the
product of a distinct gene, it more likely represents an allele of the
UbcH5B gene.
The family of structurally related E2s to which UbcH5A-C belong is believed to play key roles in the catabolism of cellular proteins. Both UbcH5A and the related AtUBC8 have been shown to catalyze the E6 and E6-AP-dependent ubiquitination of p53(30) . In in vitro studies in plant, homologues of UbcH5A-C are the most active class of E2 enzymes in crude cell extracts(24) . The importance of this E2 class is underscored by the critical role played by the ScUBC4/5, and their up-regulation in response to stress(16) . Establishing whether there are other members of this family expressed in man, and the range of substrates and E3 enzymes with which members of this family of E2s interact, are issues that await further study.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U39317 [GenBank]and U39318[GenBank].