(Received for publication, September 14, 1995)
From the
E6-AP, a 100-kDa cellular protein, was originally identified through its interaction with the E6 protein of the oncogenic human papillomavirus types 16 and 18. The complex of E6-AP and E6 specifically interacts with p53 and mediates ubiquitination of p53 in concert with the E1 ubiquitin-activating enzyme and the E2 ubiquitin-conjugating enzyme UbcH5. Recent results suggest that E6-AP is representative of a family of putative ubiquitin-protein ligases. Members of this family are characterized by a conserved C-terminal region, termed hect domain. In this paper, we describe the isolation of two human E2s, designated as UbcH6 and UbcH7, that in addition to UbcH5 can interact with E6-AP. UbcH6 is a novel member of an evolutionally conserved subfamily of E2s that includes UbcH5 and Saccharomyces cerevisiae UBC4. Although UbcH7 does not appear to be a member of this subfamily, UbcH7 efficiently substitutes for UbcH5 in E6-AP-dependent ubiquitination. Surprisingly, UbcH6 was only weakly active in this particular assay. In addition, UbcH5 but not UbcH6 or UbcH7 efficiently interacts with the hect protein RSP5. These results indicate that E6-AP can interact with at least two species of E2 and that different hect proteins may interact with different E2s.
The ubiquitin system represents a major pathway involved in
selective protein
degradation(1, 2, 3, 4) . This
pathway first requires the covalent attachment of ubiquitin, a highly
conserved 76-amino acid protein, to defined lysine residues of
substrate proteins. Ubiquitin-protein conjugates are then recognized
and degraded by a specific protease complex, the 26 S proteasome.
Protein ubiquitination involves three classes of enzymes. These are the
ubiquitin-activating enzyme E1, ()the ubiquitin-conjugating
enzymes E2, and the ubiquitin-protein ligases E3. Ubiquitin is first
activated by E1 via formation of a thioester bond between the
carboxyl-terminal glycine of ubiquitin and a cysteine residue of E1.
The activated ubiquitin is then transferred to one of a number of E2s
preserving the high energy thioester bond. The E2s have then been
thought to catalyze the final attachment of ubiquitin to a substrate
protein, often in concert with E3s. E3s have been proposed to function
by specifically binding to substrate proteins that are otherwise not
recognized by E2s. Recent results, however, suggest that at least some
E3s may also be directly involved in the final transfer of ubiquitin to
a substrate protein(5) .
Only two genes encoding proteins
with E3 activity have been cloned so far. These are UBR1 of Saccharomyces cerevisiae(6) and human
E6-AP(7) . E6-AP was originally identified through its
interaction with the E6 oncoprotein of the cancer-associated human
papillomavirus types 16 and 18(8) . The E6E6-AP complex
specifically binds to the tumor suppressor protein p53 and induces its
ubiquitination and subsequent
degradation(7, 9, 10) . An essential
intermediate step in E6-AP-dependent protein ubiquitination is the
formation of a thioester complex between ubiquitin and E6-AP (5) . Furthermore, the direction of ubiquitin transfer is from
E1 to E2 and then from E2 to E6-AP. This suggests that in this
particular system, the E3 catalyzes the final attachment of ubiquitin
to a substrate protein, rather than the E2 as previously assumed. The
cysteine residue of E6-AP involved in thioester formation has been
mapped to the carboxyl terminus. The carboxyl-terminal regions of
several proteins from different organisms show significant similarity
to the carboxyl terminus of E6-AP(5, 11) . The
cysteine residue necessary for thioester formation of E6-AP with
ubiquitin is conserved among all of these proteins. Because of this
similarity these proteins have been termed hect proteins, for
homologous to E6-AP C Terminus(11) . Although their function is
presently unknown, an intriguing possibility is that these proteins
have E3 activity, similar to E6-AP. In support of this hypothesis, it
has been shown that two of the hect proteins, namely S. cerevisiae RSP5 and a rat 100-kDa protein, can form thioester complexes with
ubiquitin(11) .
Identification of all the components
necessary and sufficient for E6E6-AP-induced ubiquitination of
p53 revealed that E6-AP interacts only with a distinct E2
activity(10) . This E2 activity is represented by members of an
evolutionally conserved subgroup of E2s that includes human UbcH5, S. cerevisiae UBC4 and UBC5, and Arabidopsis thaliana UBC8(12, 13, 14) . Recently, another
mammalian E2 activity was described that appeared to be involved in
E6
E6-AP-dependent ubiquitination of
p53(15, 16) . This E2, termed E2-F1, is different from
UbcH5, as shown by comparison of the partial amino acid sequence of
proteolytic fragments of biochemically purified E2-F1 and the sequence
of UbcH5. This indicated that there may be more than one species of E2
that can interact with E6-AP. Here we report the isolation of two human
cDNAs encoding E2s. One of these, termed UbcH6, represents a novel
member of the subfamily of E2s mentioned above. The amino acid sequence
of the other E2, termed UbcH7, includes the previously reported
sequences of the proteolytic fragments of E2-F1, indicating that UbcH7
is identical to E2-F1. In E6-AP-dependent ubiquitination, UbcH7 is as
active as UbcH5, whereas UbcH6 shows only little activity. However,
only UbcH5 interacts efficiently with RSP5. This indicates that E6-AP
can interact with different species of E2s and that different hect
proteins may interact with different E2s.
For cloning of UbcH6, two degenerate primers that were used previously to clone a cDNA encoding UbcH5 (12) were used for reverse transcription followed by PCR amplification. Reactions were performed using the RNA PCR kit from Cetus. PCR products were cloned into pGEM-1. The sequence of several clones derived from independent PCR reactions was determined using T7 DNA polymerase (Sequenase, U. S. Biochemical Corp.). The 5`-end and the 3`-end of the open reading frame were cloned using a 5`-RACE kit and a 3`-RACE kit, respectively (Life Technologies, Inc.). Reactions were performed according to the suggestions of the manufacturer. For 5`-RACE, reverse transcription was performed using a primer extending from nucleotides 516 to 497 (Fig. 1). The resulting cDNA was tailed with poly(dC) and terminal nucleotidyltransferase followed by PCR amplification. An aliquot of the PCR reaction was taken, and a second round of PCR amplification performed using a primer supplied by the 5`-RACE kit. For 3`-RACE, an oligo(dT) primer supplied by the 3`-RACE kit was used for reverse transcription followed by PCR amplification using a primer extending from nucleotides 415 to 434. An aliquot of the PCR reaction was used as a template in a second round of PCR using a primer extending from nucleotides 469 to 488. PCR products were cloned into pGEM-1 and sequenced.
Figure 1: Complete nucleotide and amino acid sequence of UbcH6 and UbcH7. The coding sequence is represented in capital letters. The previously reported peptide sequences derived from direct protein sequencing of biochemically purified E2-F1, now referred to as UbcH7, are underlined(15) . Amino acids are given in the single-letter code.
To clone UbcH7, previously
referred to as E2-F1(15) , two degenerate primers, which
correspond to two peptide sequences of UbcH7 derived from direct
protein sequencing (amino acids 106-101 and 53-59; for
numbering see Fig. 1), respectively, were used for reverse
transcription followed by PCR amplification. PCR products were cloned
into pGEM-1 and sequenced. Using a P-labeled fragment
comprising nucleotides 157 to 318 (see Fig. 1) as a probe, a
phage
clone comprising the complete open reading frame was
isolated from a random-primed cDNA library from normal human
keratinocytes (Clontech).
The complete open reading frames of UbcH6 and UbcH7 were cloned in a single fragment by reverse transcription followed by PCR amplification. For UbcH6, the primer for reverse transcription comprised nucleotides 582-563 of the open reading frame. For UbcH7, the primer for reverse transcription spanned nucleotides 465-445. Both primers contained a BamHI site at the 5`-end for cloning. For both UbcH6 and UbcH7, the opposing primer used for PCR amplification extended from nucleotides 1 to 21 of the respective open reading frame with an NdeI site at the 5`-end. The PCR products were cloned into pET-3a and sequenced.
Ubiquitination assays using GST-E6-E7 as substrate were performed as described previously(10) .
Figure 2: Comparison of amino acid sequences of UbcH6 and UbcH7 with other E2s. The amino acid sequences of UbcH6, UbcH7, UbcH5(12) , S. cerevisiae UBC4(13) , Homo sapiens HHR6A(23, 24) , and A. thaliana UBC1 (20) were aligned, and the percent similarity of each of these E2s to UbcH5 was calculated. Differences in amino acid sequence are indicated, with identities denoted by dashed lines. The active site cysteine residue is marked with an asterisk, as are the carboxyl-terminal ends of the various E2s. The amino-terminal 37 amino acids of UbcH6 are not shown (see Fig. 1).
Sequence comparison suggests that UbcH6 is related to the E2 subfamily including UbcH5 and S. cerevisiae UBC4 (Fig. 2). However, UbcH6 appears to be unique among these E2s since it contains an amino-terminal extension of approximately 40 amino acids (see Fig. 1). The functional significance of this extension is presently unknown.
Based on sequence comparison (Fig. 2), UbcH7 does not appear to be a member of the E2 subfamily mentioned above. The sequence of UbcH7 shows approximately the same similarity to UbcH5 as E2s, which are functionally not related to UbcH5 with respect to the ability to mediate E6-AP-dependent ubiquitination.
Figure 3:
Thioester adduct formation of bacterially
expressed UbcH6 and UbcH7 with ubiquitin. Thioester reactions contained P-labeled GST-ubiquitin(10) , bacterially
expressed E1(19) , ATP, and crude extracts from bacteria
harboring expression vectors encoding for different E2s as indicated.
Similar amounts of the respective E2s were used as determined by
staining with Coomassie Blue (data not shown). After 5 min at 25
°C, reactions were stopped and subjected to SDS-PAGE in the absence (A) or presence of dithiothreitol (B) followed by
autoradiography. Positions of free
P-labeled
GST-ubiquitin, of the respective E2 thioester adducts, and of molecular
size markers are indicated.
To test whether UbcH7 may indeed represent the E2 activity described by Ciechanover and colleagues(15, 16) , its ability to mediate the E6-AP-dependent ubiquitination of an E6-E7 fusion protein or of p53 was assayed. As a negative control A. thaliana UBC1 was used which was previously shown to be inactive in E6-AP-dependent ubiquitination(10) . In the presence of E6-AP, UbcH7 as well as UbcH5 could efficiently mediate ubiquitination of the E6-E7 fusion protein (Fig. 4) as well as of p53 (data not shown), with UbcH5 being slightly more active than UbcH7 under the conditions used. This strongly suggests that UbcH7 represents the E2 activity described previously.
Figure 4: Effectiveness of UbcH6 and UbcH7 in E6-AP-dependent ubiquitination. A radioactively labeled GST-E6-E7 fusion protein was used as a substrate(10) . In addition to the GST- E6-E7 fusion protein, ubiquitin-conjugation reactions contained bacterially expressed E1, ubiquitin, baculovirus-expressed E6-AP(10, 21) , and ATP. Reactions were started by the addition of the various E2s as indicated. Relative amounts of the respective E2s are given as determined by staining with Coomassie Blue. After 2 h at 25 °C, reactions were stopped, and the whole reaction mixtures were subjected to SDS-PAGE followed by autoradiography. The running position of GST-E6-E7 is indicated. The presumably monoubiquitinated form and the multiubiquitinated forms of GST-E6-E7 are marked with an arrowhead and an asterisk, respectively.
Although UbcH6 appears to be more similar to UbcH5 than UbcH7 on the amino acid sequence level, UbcH6 was only weakly active in this particular assay (Fig. 4). A possible explanation for this observation was that the amino-terminal extension of UbcH6, which distinguishes it from UbcH5 and UbcH5-related proteins of other organisms, negatively interferes with the activity of UbcH6. However, a deletion mutant of UbcH6, which initiates at the second methionine and therefore lacks the amino-terminal extension, was as inefficient in ubiquitination of the E6-E7 fusion protein as the full-length UbcH6 (data not shown).
Figure 5:
Ubiquitin thioester adduct formation of
E6-AP. Partially purified E6-AP expressed in the baculovirus system (10, 21) was incubated in the presence of E1, P-labeled GST-ubiquitin, ATP, and similar amounts of the
various E2 as indicated. After 5 min at 25 °C, reactions were
stopped in the absence (A) or in the presence of
dithiothreitol (B). The whole reaction mixtures were separated
by SDS-PAGE at 4 °C followed by autoradiography. The running
positions of the ubiquitin thioester adducts of the respective E2s and
of E6-AP are indicated as well as the running positions of molecular
size markers.
Recently it was reported that UbcH5 can also
interact with RSP5, a member of the hect family of proteins, as
measured by UbcH5-dependent formation of ubiquitin thioester adducts of
a deletion mutant of RSP5 lacking the carboxyl-terminal 6 amino acids
(RSP5C6). It was therefore tested whether UbcH7 or UbcH6, or both,
could substitute for UbcH5 in this reaction (Fig. 6). In
contrast to the experiments with E6-AP, both UbcH6 and UbcH7 were
significantly less active than UbcH5, as judged by the appearance of
thioester complexes of RSP5
C6 with ubiquitin.
Figure 6:
Ubiquitin thioester adduct formation of
RSP5C6. A deletion mutant of RSP5 lacking the carboxyl-terminal 6
amino acids (RSP5
C6) was expressed in the baculovirus system as
described previously(11) . Thioester complex formation of
RSP5
C6 with ubiquitin was assayed as described in Fig. 5. A, reactions were terminated in the absence of dithiothreitol; B, reactions were terminated in the presence of 100 mM dithiothreitol. Running positions of the ubiquitin thioester
adducts of the different E2s and of RSP5
C6, respectively, are
indicated as well the running positions of molecular size
markers.
The ubiquitin system plays a major role in selective protein degradation(1, 2, 3, 4) . Selective protein degradation requires the specific targeting of many different proteins, often at particular stages of the cell cycle or differentiation. The selectivity of the ubiquitin system appears to be mediated by E2s, often in conjunction with E3s. It was previously shown that E6-AP and RSP5, two members of the hect family of putative E3 proteins, interact specifically with a subgroup of human E2s represented by UbcH5(11, 12) . We have now cloned two additional human E2s, designated UbcH6 and UbcH7, that can interact with E6-AP and RSP5. However, whereas RSP5 interacts only weakly with UbcH6 and UbcH7 compared with UbcH5, UbcH7 is as active as UbcH5 in E6-AP-dependent ubiquitination. This indicates that different hect proteins may require different E2 activities for protein ubiquitination. The differential interaction between E3s and E2s may further contribute to ensure the high specificity of protein ubiquitination.
UbcH6 was cloned by PCR using degenerate
oligonucleotide primers that correspond to highly conserved regions of
a subfamily of E2s, which includes UbcH5 and S. cerevisiae UBC4 and UBC5(11, 13) . Accordingly, amino acid
sequence comparison with other E2s revealed that UbcH6 has the highest
similarity to the members of this subfamily. Nevertheless, UbcH6
appears to be a unique member of this subfamily in that it contains an
amino-terminal extension of approximately 40 amino acids, in contrast
to all other members of the subfamily described to
date(11, 13, 14, 31, 32) .
Despite the similarity, UbcH6 could not efficiently substitute for
UbcH5 or S. cerevisiae UBC4 in E6-AP-dependent protein
ubiquitination. It seemed possible that the amino-terminal extension
masks a domain of UbcH6 necessary for interaction with E6-AP or induces
a conformation of UbcH6, which is not properly recognized by E6-AP.
However, a deletion mutant of UbcH6 lacking the amino-terminal
extension was not more efficient in E6-AP-dependent ubiquitination than
the full-length protein. The amino-terminal extension contains an
unusual high number of serines. Therefore, it is conceivable that
modification of the amino-terminal extension, such as phosphorylation,
may result in a form of UbcH6 that, like UbcH5, can efficiently
interact with E6-AP. Alternatively, UbcH6 and UbcH5 may interact with a
different set of E3s. In support of the latter hypothesis is the recent
observation by S. Jentsch and co-workers ()that a murine E2,
which is almost identical to UbcH6 at the amino acid sequence level,
could only partially substitute for S. cerevisiae UBC4 and
UBC5 in genetic experiments.
UbcH7 was cloned based on the published
sequences of proteolytic fragments derived from a chromatographically
purified E2 activity termed E2-F1(15) . Similar to UbcH5, E2-F1
was reported to function in E6-AP-dependent
ubiquitination(16) . Since UbcH5 and E2-F1 have similar
chromatographic properties, ()however, it seemed possible
that the E2-F1 preparation used was contaminated by UbcH5. This
possibility can now be excluded since bacterially expressed UbcH7 was
indeed functional in E6-AP-dependent ubiquitination. Amino acid
sequence comparison of UbcH7 with UbcH5 suggests that UbcH7 is not more
related to UbcH5 than E2s, which are inactive in E6-AP-dependent
ubiquitination. This indicates that E6-AP can interact with different
species of E2s. Whether there are additional E2s that may interact with
E6-AP is not clear at present. Such E2s, however, should have
chromatographic properties similar to UbcH5 and UbcH7 because only
fractions obtained by cation-exchange chromatography of cellular
extracts that contain UbcH5 and UbcH7 can support E6-AP-dependent
ubiquitination(10) .
An intriguing but purely
speculative hypothesis is that in the presence of UbcH7, E6-AP has an
altered substrate specificity from that in the presence of UbcH5.
However, although E6-AP interacts with both UbcH5 and UbcH7 in in
vitro systems, further experiments will be necessary to determine
whether these interactions also exist in vivo.
Similar amounts of thioester complexes of E6-AP and ubiquitin were detected in the presence of UbcH5 and UbcH7, respectively. In contrast, transfer of ubiquitin to RSP5 was much less efficient in the presence of UbcH7 than in the presence of UbcH5. The reason for this apparent difference between RSP5 and E6-AP is not known. As mentioned above, UbcH7 is apparently not a member of the UbcH5-like subfamily of E2s. Nevertheless it seems likely that the regions (or structures) of UbcH5 and UbcH7 that are necessary for interaction with E6-AP and RSP5 are similar to each other. Construction of deletion and point mutants as well as of chimeric proteins of UbcH5 and UbcH7 or UbcH6, respectively, should allow definition of those regions.
Both E6-AP and RSP5 are
members of the family of hect proteins, which are putative
E3s(11) . E3s are presumably involved in mediating the
substrate selectivity of protein ubiquitination, indicating that there
may be a large number of proteins with E3-like activities. Recent data
base searches suggest that human cells encode for 10 or more hect
proteins. Assuming that all of these proteins interact with
the ubiquitin system, it will be interesting to see whether hect
proteins in general share the property of E6-AP and RSP5 to interact
with members of the UbcH5-like subfamily of E2s or whether some of
these may interact with other E2s. Characterization of hect proteins
and their interplay with E2s should contribute to define the precise
role of E2s and E3s in mediating substrate recognition in E3-dependent
protein ubiquitination.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X92962 [GenBank]and X92963[GenBank].