(Received for publication, February 28, 1997, and in revised form, April 7, 1997)
From the Laboratory of Immune Cell Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-1152
In most instances, the transfer of ubiquitin to target proteins is catalyzed by the action of ubiquitin protein ligases (E3s). Full-length cDNAs encoding murine E6-associated protein (mE6-AP) as well as Nedd-4, a protein that is homologous to E6-AP in its C terminus, were cloned. Nedd-4 and mouse E6-AP are both enzymatically active E3s and function with members of the UbcH5 family of E2s. Mouse E6-AP, like its human counterpart, ubiquitinates p53 in the presence of human papilloma virus E6 protein, while Nedd-4 does not. Consistent with its role in p53 ubiquitination, mE6-AP was found both in the nucleus and cytosol, while Nedd-4 was found only in the cytosol. Binding studies implicate a 150-amino acid region that is 40% identical between mE6-AP and Nedd-4 as a binding site for the C-terminal portion of an E2 enzyme (UbcH5B). Nedd-4 was determined to have a second nonoverlapping E2 binding site that recognizes the first 67 amino acids of UbcH5B but not the more C-terminal portion of this E2. These findings provide the first demonstration of physical interactions between mammalian E2s and E3s and establish that these interactions occur independently of ubiquitin and an intact E3 catalytic domain. Furthermore, the presence of two E2 binding sites within Nedd-4 suggests models for ubiquitination involving multiple E2 enzymes associated with E3s.
The conjugation of proteins to ubiquitin (Ub)1 followed by their targeted degradation in the 26 S proteasome constitutes a central means of effecting regulated protein degradation in eucaryotic cells (reviewed in Refs. 1-8). Ubiquitination is implicated in the proteasomal degradation of a rapidly growing list of regulatory proteins including cell cycle regulators, transcription factors, kinases, phosphatases, and tumor suppressors. Additionally, there are recent data suggesting a role for this post-translational modification in receptor-mediated endocytosis (8-10).
Ub is a highly conserved 76-amino acid polypeptide that is found
covalently bound to target proteins as monomers or chains. This occurs
as the result of a multienzyme process involving sequential thiol-ester
bonds with classes of enzymes termed E1-E3. This cascade culminates in
the formation of isopeptide bonds between the C terminus of Ub and the
-amino groups of lysines on either a substrate or the growing end of
a protein-bound multiubiquitin chain. This process is initiated by the
ATP-dependent formation of high energy thiol-ester bonds
between E1 (Ub-activating enzyme) and the C terminus of Ub. Ub is then
transferred to E2s (UBCs or Ub-conjugating enzymes). Models based on
the N-end rule E3s of yeast and mammals (1, 2) had E3s functioning
primarily as docking proteins that recognize substrates and Ub-bound
E2s, facilitating the transfer of Ub from E2 to substrate. However,
more recent findings obtained with E6-AP (E6-associated protein) have
demonstrated that, at least in some cases, Ub moieties bound to E3s
through thiol-ester linkages represent the final intermediates in the
ubiquitination cascade (11, 12). It remains to be determined whether
the N-end rule E3s similarly form thiol intermediates with Ub.
Only one E1 enzyme has been characterized in mammals. In contrast, over 10 distinct E2s have been characterized in humans, as well as in yeast, and in the plant Arabidopsis thaliana (reviewed in Refs. 4 and 13). Since substrate specificity for ubiquitination appears to lie largely at the level of E3-substrate interactions, it is generally believed that there are a large number of E3s. This theory is supported by the partial purification of multiple distinct E3 activities from mammalian cells and other higher eucaryotes (Refs. 14-20; reviewed in Ref. 8). cDNAs encoding three yeast E3s have been characterized (21-23). However, human E6-AP is the only characterized mammalian E3 for which the primary amino acid sequence is known (24). This E3 catalyzes p53 ubiquitination but does so only in the presence of viral E6 proteins from strains of human papilloma virus (HPV) that are high risk for the development of uterine cervical carcinomas (HPV-16 and HPV-18). E6-dependent ubiquitination is presumably responsible for the diminished levels of p53 characteristic of HPV-16- and HPV-18-infected cells (24, 25). No other cellular substrates for E6-AP have been identified.
The characterization of E6-AP led Huibregtse et al. (12) to
identify several cDNAs in GenBankTM with deduced
protein sequences homologous to the C terminus of E6-AP; they termed
these regions HECT (homology to E6-AP
carboxyl terminus) domains. The sine qua
non of a HECT domain is a conserved cysteine residue that forms a
thiol-ester with Ub. Among the HECT-encoding cDNAs was a single
partial mouse cDNA termed Nedd-4, first identified by its
differential expression in fetal relative to adult brain (26). Recently
rat Nedd-4 was cloned as a binding partner for the subunit of the
amiloride-sensitive epithelial sodium channel using the yeast
two-hybrid system (27). This association occurs through interactions of
tryptophan-containing WW domains of Nedd-4 with proline-rich PY motifs
within sodium channel subunits (27-29). In Liddle's syndrome, an
autosomal dominant form of hypertension characterized by increased
activity and cell surface expression of sodium channels, mutations
within genes encoding subunits of the sodium channel result in loss of
PY motifs (30). This raises the intriguing possibility that this ion
channel is regulated by Nedd-4-dependent ubiquitination.
However, until now Nedd-4 has not been demonstrated to be a
catalytically active E3. In the present study, we report the cloning of
cDNAs encoding full-length murine Nedd-4 and murine E6-AP (mE6-AP)
and determine that they are both catalytically active E3s. In addition,
their subcellular localizations and their physical interactions with an
E2 enzyme are investigated.
Probes for
screening cDNA libraries were generated from first strand cDNA
from Jurkat cells or mouse brain using a cDNA cycle kit
(Invitrogen, San Diego, CA) followed by PCR amplification using
oligonucleotide primers2 for 35 cycles with
the following parameters: 94 °C for 1 min, 55 °C for 1 min, and
72 °C for 1 min, with a final 10-min extension at 72 °C. Probes
corresponded to bases 1991-2572 of human E6-AP (24) and bases
1489-2097 of the partial mouse Nedd-4 sequence (26). For both mE6-AP
and Nedd-4, 1 × 106 independent phage plaques of an
oligo(dT)-primed ZAP (Stratagene) mouse thymus cDNA library were
screened using 32P-labeled random primed probes (31).
Replica filters were hybridized at 55 °C for 24 h in 6 × SSC, 5 × Denhardt's solution, 0.5% SDS, 10 mM EDTA,
100 µg/ml of salmon sperm DNA. Filters were subject to a final wash
in 0.2 × SSC and 0.1% SDS at 55 °C. Positive clones were
subcloned into pBluescript SK
(Stratagene, La Jolla, CA)
by in vivo excision (Stratagene), and sequenced. Alignments
were performed using the BESTFIT program through the National Library
of Medicine.
Bacterial
lysates expressing recombinant E2s were prepared as described (32).
Plasmid encoding nUbcH5B (amino acids 1-67 of UbcH5B) was
generated by PCR amplification using UbcH5B as template and ligated
into pET-15b (Novagen). Plasmid encoding cUbcH5B (amino
acids 67-143 of UbcH5B) was generated by PCR using UbcH5B as template,
with sense and antisense nucleotides that created a 5 NcoI
site and a 3
XhoI site. The product was cloned into
pBluescript, digested with SalI and PstI, and
subcloned into pMAL-C2 (New England Biolabs) to generate plasmid
encoding MBP-cUbcH5B. Purified plasmids were transfected
into BL21(DE3) Escherichia coli (Novagen), and induction of
recombinant protein with isopropyl-
-D-thiogalactoside (Life Technologies, Inc.) was carried out per the manufacturer's instructions. E. coli extracts were prepared from frozen
cell pellets as described previously (32). Protein content of crude extracts were estimated by Coomassie Blue staining.
To generate a plasmid encoding Schistosoma japonicum
glutathione S-transferase (GST) fused to amino acids 32-869
of mE6-AP, mE6-AP cDNA in pBluescript was digested with
HincII and SmaI and ligated into the
SmaI site of pGEX-KG (33) (pGEX-mE6-AP). To generate
pGEX-mE6-AP432 and pGEX-mE6-AP274, pGEX-mE6-AP was digested with
XbaI or XhoI respectively and autoligated.
pGEX-mE6-AP679 was generated by digestion of pGEX-mE6-AP with
BglI and HindIII, filling in with Klenow, and
autoligation. For GST-Nedd-4, mouse Nedd-4 cDNA in pBluescript was
digested with AvrII and SpeI and ligated into the
XbaI site of pGEX-KG (pGEX-Nedd-4). Plasmid encoding GST-Nedd-4-N (amino acids 52-777) was constructed by digestion of
pGEX-Nedd-4 with HindIII and self-ligation. GST-Nedd-4-C was generated from pBS-Nedd-4-15 by digestion with HindIII and
ligating the fragment (bases 1876-3480) into the HindIII
site of pGEX-KG. Plasmid encoding GST-Nedd-4CT (amino acids 423-670)
was generated by PCR using Nedd-4 cDNA as a template and a sense
primer corresponding to bases 1876-1895 with an EcoRI site
and an antisense primer corresponding to bases 2604-2621 with a stop
codon and an XhoI site. The product was subcloned into
pBluescript II, sequenced, and subcloned into pGEX-KG in the
EcoRI and XhoI sites. GST fusion proteins were
expressed in E. coli strain DH5 induced with 0.1 mM isopropyl-
-D-thiogalactoside. Bacterial
cell pellets resuspended in phosphate-buffered saline were lysed by
sonication, and cellular debris was removed by centrifugation at
13,000 × g. Glutathione-Sepharose (Pharmacia Biotech
Inc.) was added to the supernatant, and the mixture was rotated at
4 °C overnight. Beads were washed in phosphate-buffered saline and
either stored in this buffer or eluted in 50 mM Tris (pH
8.0) and 10 mM reduced glutathione. Protein content was
estimated by Coomassie Blue staining.
Reaction mixtures containing 0.5 µg
of purified GST fusion proteins and 1 µl of crude lysate from
E. coli expressing wheat E1 (34), 1 µl of the appropriate
E2s, and 10 µg of bovine Ub (Sigma) were incubated as described (32)
for 2 h at 30 °C, followed by the addition of
SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer containing
4% -mercaptoethanol (reducing sample buffer). Samples were resolved
by SDS-PAGE, transferred to membranes, and immunoblotted with
previously described affinity-purified anti-Ub raised against Ub
coupled to chicken serum albumin (35) followed by development with
125I-protein A (ICN, Costa Mesa, CA) and visualized by
autoradiography as described (35). In these assays, bacterial proteins
from the crude extracts serve as the source of ubiquitination
substrates. For p53 ubiquitination reactions, RNA was prepared from
plasmids encoding HPV-E6 and human p53 (36) using Ribomax (Promega,
Madison, WI), and in vitro translation was carried out in
wheat germ extract (Promega) without (E6) or with (p53)
[35S]methionine (ICN). Reaction mixtures containing E1
and E2s with either 0.5 µg of crude lysate from SF9 cells transfected
with baculovirus expressing a 95-kDa truncated human E6-AP (a gift from
J. Huibregtse and P. Howley) or 0.5 µg of purified GST-E3 fusion
proteins were incubated as above prior to resolution on 12% SDS-PAGE
and autoradiography.
For
Myc-tagged mE6-AP, a cDNA fragment encoding an
NH2-terminal Myc tag and a four-amino acid linker (37) in
continuity with the NH2 terminus of mE6-AP (up to base 347)
was amplified from pBS-E6-AP-11 using oligonucleotides that resulted in
the generation of a 5 KpnI site. The product was digested
with KpnI and HincII and ligated into
pBS-E6-AP-11 that was similarly digested. The resultant product was
digested with KpnI and SmaI and ligated from
KpnI to EcoRV in pcDNA3 (Invitrogen) to
generate pcDNA-Myc-E6-AP.
For HA-tagged-Nedd-4, the vector pSNeo was first generated. pcDNA3
was digested with BglII and SacI, a 4.4-kilobase
pair fragment purified and filled in by T4 polymerase. pSX-SR (38)
was digested with SalI, and a 2.2-kilobase pair fragment was
purified and filled in by Klenow. These fragments were ligated to
generate pSNeo. A cDNA fragment encoding Nedd-4 with an HA-tagged
sequence at its N terminus was amplified from pBS-Nedd-4-15 plasmid by
PCR using a 5
primer that generated a NotI site followed by
an HA tag contiguous with the amino terminus of Nedd-4 and an antisense oligo that included the AvrII site of Nedd-4. The product
was digested with NotI and AvrII, purified, and
ligated into pBS-Nedd-4-15 that had been similarly digested. This
product was digested with NotI and EcoRI and
subcloned into pSneo from NotI to EcoRI to generate pS-HA-Nedd-4.
Cells transfected 48 h earlier with 20 µg of DNA were fractionated basically as described by Grenfell (39). Nuclear and cytosolic fractions were resolved on SDS-PAGE followed by immunoblotting with the appropriate antibodies. Immunofluorescence staining of transfected COS-7 cells was carried out as described (40) using culture supernatants from cells producing either 9E10 (anti-Myc) (41) or 12CA5 (anti-HA) (Boehringer Mannheim).
Binding AssayBinding assays were performed by a modification of the method of Huibregtse et al. (25). Briefly, 1 µg of recombinant E2 enzyme was mixed with 1 µg of GST fusion proteins immobilized on glutathione-Sepharose in 400 µl of binding buffer (25 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5% Nonidet P-40, and 5 mM dithiothreitol). Mixtures were rotated at 4 °C for 20 h, washed four times with binding buffer, and, after elution in reducing sample buffer, resolved by SDS-PAGE. Proteins were transferred to Immobilon-P (Millipore Corp.) and reacted with affinity-purified anti-Caenorhabditis elegans UBC2 polyclonal antibody (1 µg/ml) (42) or with anti-MBP polyclonal antibody (1:10,000 dilution) (New England Biolabs). Samples were then treated as described for anti-Ub immunoblotting (35).
Probes based on human E6-AP and C-terminal nucleotide
sequences from the partial murine Nedd-4 were used to isolated
full-length cDNAs encoding mouse E6-AP and Nedd-4 from a mouse
thymocyte library. mE6-AP has a predicted molecular mass of 99.4 kDa
and a pI of 4.74 and exhibits 96.2 and 94.2% deduced amino acid
similarity and identity, respectively, with human E6-AP (Fig.
1A). Unlike human E6-AP, an ATG in the
context of a Kozak sequence (43) was identified within the murine E6-AP
cDNA. In the 18-amino acid region (amino acids 396-413) implicated
in the binding of HPV E6 to human E6-AP (25), a single conservative
amino acid difference was found (Glu Asp).
The full-length murine Nedd-4 (Fig. 1B) has a predicted molecular weight of 90.6 kDa and an estimated pI of 5.46. The full-length murine Nedd-4 clone exhibits 95.9% identity and 97.7% similarity with the rat Nedd-4 and shows 95.2% similarity and 90.1% identity with an unpublished GenBankTM-registered human Nedd-4. The mouse, as well as the rat, Nedd-4 cDNA has a 67-amino acid gap relative to the deduced amino acid sequence of the human protein; this gap corresponds to one of the four WW domains in human Nedd-4. To determine if this difference is due to alternative splicing in the mouse, PCR amplification of first strand cDNA from multiple mouse tissues was performed across this region; however, no evidence of a longer product was found (not shown). Messenger RNA for both mE6-AP and p53 was found in all murine tissues examined. Mouse E6-AP mRNA was most abundant in brain, thymus, and heart, while Nedd-4 was found at the greatest levels in lung and kidney (not shown).
mE6-AP and Nedd-4 are Ubiquitin Protein Ligases That Function with the Same Family of E2sTo determine whether mE6-AP and Nedd-4
function as E3s, recombinant GST-E6-AP (amino acids 32-869 of mE6-AP)
and GST-Nedd-4 (amino acids 52-777 of Nedd-4) were generated and
assessed for their ability to catalyze the ubiquitination of bacterial
cellular proteins (Fig. 2, A and
B). Since multiple proteins serve as substrates in this
assay, ubiquitinated species generally appear as a smear on anti-Ub
immunoblots (32). In these experiments, wheat E1 (34) and a human E2
(UbcH5B) that functions with human E6-AP were utilized (32). As shown
(Fig. 2A), Nedd-4 catalyzes ubiquitination in the presence
of E1 and E2. Some weak E2-independent, E1-dependent Nedd-4-mediated ubiquitination is also observed. The GST fusion with
mE6-AP (Fig. 2B) also exhibited a low level of
E2-independent activity that was substantially enhanced by the addition
of UbcH5B. We consistently observed that GST-E6-AP was less active than
GST-Nedd-4. Whether this reflects differences in the activity of these
two proteins, reflects issues of substrate specificity, or is a
manifestation of differences in folding of these recombinant fusion
proteins remains to be determined. While other studies have
demonstrated thiol-ester formation involving HECT family proteins (11,
12), this provides the first evidence that a mammalian HECT family protein other than human E6-AP functions as a Ub protein ligase.
Members of the UbcH5 family of E2s, which are over 87% identical to each other, and a closely related A. thaliana E2, AtUBC8, all function with human E6-AP in protein ubiquitination, while a more distantly related E2, AtUBC1 does not (32, 44, 45). To determine whether Nedd-4 and mE6-AP exhibit specificity at the level of E2 interactions, equivalent amounts of thiol-ester-forming activity for each of these E2s were estimated as described (32) (not shown) and utilized in ubiquitination assays (Fig. 2, C and D). For both Nedd-4 and mE6-AP, UbcH5 family members and the closely related AtUBC8 all facilitated ubiquitination of bacterial cellular proteins, while AtUBC1 showed minimal or no activity. As expected, when p53 was used as a substrate in ubiquitination assays, mE6-AP catalyzed p53 ubiquitination in an E6-dependent manner, while Nedd-4 did not catalyze p53 ubiquitination either with or without added E6 (not shown).
Differential Subcellular Localization of E6-AP and Nedd-4mE6-AP and Nedd-4 both manifest potential nuclear
localization signals defined by the presence of clusters of basic amino acids (amino acids 413-417 of mE6-AP and amino acids 402-413 of Nedd-4) (46). To assess subcellular localization, constructs encoding
full-length mE6-AP with an N-terminal Myc tag and full-length Nedd-4
with an HA tag were generated. Analysis of transfected COS-7 cells by
immunofluorescence suggests a mixed nuclear-cytosolic distribution for
mE6-AP. In contrast, Nedd-4 exhibits a pattern consistent with
distribution in the cytosol and not the nucleus (Fig.
3). To substantiate these observations, nuclear and
cytosolic fractions were prepared from transfected COS-7 cells and
analyzed by immunoblotting (Fig. 4). In accord with the
immunofluorescence results, Nedd-4 was found only in the cytosol (Fig.
4, lanes 9 and 10), while mE6-AP was detected
both in the nucleus and cytosol (Fig. 4, lanes 3 and
4). A Myc-tagged cytosolic protein tyrosine kinase, ZAP-70
(47, 48), employed as a control, demonstrated a cytosolic distribution
pattern. The finding of Nedd-4 in the cytosol is consistent with a
putative role in regulating sodium channel activity (27), while the
presence of nuclear mE6-AP is in accord with an in vivo role
for this protein in catalyzing E6-mediated p53 ubiquitination (49).
Interactions between Murine E3s and UbcH5B
To evaluate the
molecular basis for interactions between E2s and E3s, the binding of
recombinant UbcH5B-derived proteins to GST-E3 fusion proteins was
assessed (see Fig. 5 for schematic representation of
constructs and Table I for summary of binding studies).
GST-E6-AP was incubated with either full-length UbcH5B or an equivalent
amount of a truncated UbcH5B containing only the first 67 amino acids
of this E2 (nUbcH5B). Bound material was detected by
immunoblotting with an antisera that recognizes the NH2
terminus of UbcH5B (Fig. 6A). UbcH5B bound
specifically to GST-E6-AP; however, no specific binding of
nUbcH5B was detected. To detect associations between the
carboxyl half of UbcH5B and GST-E6-AP, a chimeric protein was generated
in which the maltose-binding protein (MBP) was placed upstream of amino
acids 68-147 of UbcH5B (MBP-cUbcH5B).
MBP-cUbcH5B bound GST-E6-AP, while MBP itself bound to
neither GST alone nor to GST-E6-AP (Fig. 6B). To further
characterize this association, truncated forms of mE6-AP were
generated. UbcH5B and MBP-cUbcH5B bound to a GST fusion
protein in which the 190 C-terminal amino acids of E6-AP, including the
region implicated in catalytic activity, were deleted (GST-E6-AP679)
(Fig. 6C, lane 3). More severely truncated
fusions of GST with mE6-AP bound neither UbcH5B nor
MBP-cUbcH5B (Fig. 6C).
|
In contrast to E6-AP, the amino (nUbcH5B) and carboxyl
(MBP-cUbcH5B) halves of UbcH5B as well as full-length UbcH5B
specifically bound GST-Nedd-4 (Fig. 7A,
lanes 6 and 10, and Fig. 7B,
lane 6). These associations were further evaluated using
constructs encoding GST fusions with either amino acids 52-422 of
Nedd-4 (GST-Nedd-4-N) or amino acids 423-777 of Nedd-4 (GST-Nedd-4-C).
UbcH5B bound to both of these regions of Nedd-4 (Fig. 7A,
lanes 7 and 8), suggesting the existence of at
least two E2 binding sites within Nedd-4. Confirmation of these dual E2
binding sites comes from the findings that nUbcH5B bound to
GST-Nedd-4 and GST-Nedd-4-N but not to GST-Nedd-4-C (Fig.
7A, lanes 10-12), while MBP-cUbcH5B
associated specifically with GST-Nedd-4 and GST-Nedd-4-C but not
GST-Nedd-4-N (Fig. 7B, lanes 6-8). Alignment of
the E6-AP and Nedd-4 constructs that bind MBP-cUbcH5B
implicate a region corresponding to amino acids 521-679 of mE6-AP and
423-583 of Nedd-4 as a site of interaction. Further confirmation of
the ability of this region to bind the C-terminal half of UbcH5B was
provided by the generation of a truncated form of GST-Nedd-4-C in which
the C-terminal 116 amino acids, including the highly conserved region
implicated in catalytic activity of HECT family E3s, was deleted
(GST-Nedd-4-CT). As shown (Fig. 7C), both the full-length
(UbcH5B) and the C-terminal (MBP-cUbcH5B) proteins bound to
GST-Nedd-4-CT (amino acids 423-670 of Nedd-4), while
nUbcH5B did not.
These results establish the existence of a discrete, conserved site of interaction between the C terminus of UbcH5B and at least two members of the HECT family of E3s. This binding requires neither Ub, E1, nor an intact E3 catalytic domain. Because GST-Nedd-4-C contains both an E2 binding site and the portion of Nedd-4 implicated in the catalytic activity of HECT family E3s, we evaluated whether this fusion protein has the ability to catalyze the ubiquitination of bacterial cellular proteins. Neither GST-Nedd-4-C nor GST-Nedd-4-N had detectable catalytic activity (Fig. 7D). Thus, while an E2 binding site and the conserved catalytic domain are found within the C-terminal half of Nedd-4, additional sequences, possibly including the second E2 binding site, are required for Nedd-4 to function as a ubiquitin protein ligase.
We have cloned and characterized two widely expressed murine ubiquitin protein ligases that interact with a family of core E2 enzymes. Consistent with an in vivo role in p53 ubiquitination, mE6-AP is found in the nucleus as well as the cytosol. We have determined that Nedd-4, first identified as a partial cDNA differentially expressed in developing brain (26), also functions as a Ub protein ligase. Nedd-4 shows no activity toward p53, and, in contrast to mE6-AP, Nedd-4 was not detected in the nucleus.
The only previous demonstration of a physical interaction between an E2 and E3 came from work on UBR1, the N-end rule E3 of S. cerevisiae (50). In that study, UBR1, which is structurally unrelated to Nedd-4 and E6-AP, co-purified with ScUBC2 when yeast lysates expressing each of these enzymes were mixed. We have established the existence of two E2 binding sites within Nedd-4. One of the two sites within Nedd-4 binds the C-terminal half of UbcH5B (cUbcH5B), while the other binds the N-terminal half of this E2 (nUbcH5B). A cUbcH5B binding site was also found within mE6-AP, and while no association between nUbcH5B and mE6-AP was found, this does not preclude a physiologically relevant interaction not detectable by in vitro binding. Since all of the proteins used in these studies were expressed in E. coli, these E2-E3 interactions do not require Ub, E1, or other known components of the Ub-conjugating system.
When mE6-AP and Nedd-4 are aligned, the region common to all of the
recombinant proteins that bind cUbcH5B (amino acids 522-680 of mE6-AP; 423-582 of Nedd-4) exhibits 40% identity (Fig.
8). Notably, this region does not encompass the
C-terminal 100 amino acids of these E3s, which includes the catalytic
cysteine. Three other HECT family proteins, a rat 100-kDa protein,
S. cerevisiae Rsp5, and Schizosaccharomyces pombe
Pub1 have been demonstrated to form thiol-ester bonds with Ub (11, 12,
22). Two of these, Rsp5 and Pub1, have been shown to be enzymatically
active E3s (12, 22). When mE6-AP, Nedd-4, Rsp5, and Pub1 are aligned (Fig. 8), the region implicated in binding cUbcH5B
demonstrates 27% identity, not substantially different from the
C-terminal 100 amino acids that surround the catalytic cysteine (30%
identity). By comparison, the more NH2 regions of these
four E3s exhibit no significant identity. While the rat 100-kDa protein
also exhibits substantial homology with Nedd-4 and mE6-AP in the region
implicated in E2 binding, this rat protein also has insertions of 9, 32, and 8 amino acids in this part of the protein (not shown). Since there is little information available on this rat protein other than
the fact that it forms E2-dependent thiol-esters with Ub, it is premature to speculate on the significance of these differences. Notably, UbcH7 (also known as E2-F1 (51)), which is only 65% homologous to the UbcH5 family of E2s, functions with human E6-AP but
not with Rsp5 in in vitro assays (23). It will be of
interest whether the region of interaction with UbcH7 is distinct from, or overlaps, the cUbcH5 binding site.
As already alluded to, Nedd-4 also manifests a second E2 binding site.
This binding site for nUbcH5B is located between amino acids
55 and 422 of Nedd-4. While it is possible that in vivo one
E2 molecule binds Nedd-4 through two distinct contact points, the fact
that the interactions of GST-Nedd-4 fusion proteins with cUbcH5B and nUbcH5B are each of sufficient
strength to be detected in an in vitro binding assay makes
it likely that Nedd-4 has the capacity to simultaneously bind two E2
molecules. The existence of these two binding sites within an E3 is
complemented by genetic studies in S. cerevisiae, where
multiple E2s participate in the ubiquitination of the MAT2 repressor
(52), by findings from the yeast two-hybrid system indicating hetero-
and homotypic interactions of E2 molecules (52, 53), and by
cross-linking studies (54). Taken together with our observations, these
findings indicate that E2-E2 interactions may occur in the context of
E3-containing complexes and open for consideration models in which
multiple E2s cooperate in the generation of protein-bound chains of
Ub.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U96636[GenBank] (murine E6-AP) and U96635[GenBank] (murine Nedd-4).
We thank J. S. Bonifacino, E. P. M. Candido, P. Howley, J. Huibregtse, L. E. Samelson, R. Wange, M. Yang, R. D. Vierstra, and M. Zhen for reagents. We also thank S. Tiwari, J. D. Weissman, and C. M. Zacharchuk for helpful comments on this manuscript.