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INTRODUCTION |
Targeted protein degradation in all eukaryotic cell types is
carried out by the ubiquitin-26 S proteasome pathway (1, 2). The covalent attachment of ubiquitin to target proteins involves an
energy-dependent, multistep pathway. Ubiquitin, activated
in the presence of ATP by an ubiquitin-activating enzyme, is
transferred to an ubiquitin-conjugating enzyme
(E2)1 by trans-thiol
esterification. It is then transferred to
-amino groups of lysine
residues in target proteins, generally with the aid of an
ubiquitin-protein ligase (E3) and sometimes involving an additional
protein, the multiubiquitin chain assembly factor (3). The conjugated
ubiquitin molecule can itself serve as an ubiquitylation substrate, and
repeated ubiquitylation leads to the formation of poly(ubiquitin)
chains. Deubiquitylating enzymes may remove ubiquitin molecules before
a sufficiently large branch structure has been synthesized to activate
26 S proteasome-mediated protein destruction.
The specificity of protein targeting by the ubiquitylation pathway lies
in the unique interaction between a particular combination of an E2, an
E3, and a target protein (4-6). E2s have been relatively easy to
identify as a result of their strong structural homologies. By
contrast, the identification of E3 families has been more difficult because of their more complex, multicomponent nature. We employed a
yeast two-hybrid approach to isolate potential E3 components that
interact with the human E2, UbcH7. This identified two proteins that we
termed HHARI (human homologue of
Drosophila ariadne) and H7-AP1
(UbcH7-associated protein 1) (7).
These proteins also bound to UbcH8 in in vitro assays. As
observed with many components of the ubiquitin pathway, both UbcH7 and
HHARI are structurally and functionally highly conserved throughout
evolution (8-14). Indeed, an interaction between orthologues of UbcH7
and HHARI has subsequently been demonstrated in both mouse (12) and
Drosophila (14).
HHARI and H7-AP1 are characterized by the presence of RING
(really interesting new
gene) finger domains, which apparently regulate their
interaction with UbcH7. The RING finger domain is a Cys/His-rich,
zinc-chelating domain that promotes both protein-protein and
protein-DNA interactions and is defined as
Cys1-X2-Cys2-X9-39-Cys3-X1-3-His4-X2-3-(Cys/His5)-X2-Cys6-X4-48-Cys7-X2-Cys8,
where X can be any amino acid (15). Proteins containing RING finger motifs are further subgrouped depending on whether a Cys or His
residue is found at Cys/His5 within the motif, so that they
are defined as either RING-HC (Cys5) or a RING-H2
(His5) type. Proteins containing RING finger domains
represent a large group with diverse cellular functions. These include
roles in apoptosis, cell cycle control, and prevention of oncogenesis. More recently, however, an increasing number of proteins containing RING finger domains have been implicated in the ubiquitylation pathway
(see reviews in Refs. 16-18), with many displaying E3 ligase activity
in in vitro assays. These proteins can act either alone (19-22) or as components of multiprotein E3 ligases such as the Skp·Cdc53·F-box complexes (23, 24), the anaphase promoting complex
(25, 26), or the von Hippel-Lindau protein complex (27, 28).
It has become evident that HHARI is a member of a family of RING-HC
finger UbcH7-binding proteins that also includes c-CBL and Parkin, a
protein mutated in autosomal dominant juvenile onset Parkinson's
disease (Ref. 29; Online Mendelian Inheritance in Man number
602544). HHARI and Parkin both contain two RING-HC type domains
separated by an additional Cys/His-rich region, which has been termed
either the IBR (in between RING
fingers) (30) or DRIL (double RING finger
linked) (31) domain (see Fig. 6A). This R-IBR-R
domain structure regulates these two proteins interactions with
UbcH7 (7, 12, 14, 22). Disease-associated mutations within the R-IBR-R
domain of Parkin prevent its interaction with UbcH7 and destroy E3
activity (22). Kahle et al. (32) have recently designated
these proteins as PAUL (Parkin/Ariadne-like ubiquitin ligase) proteins. The proto-oncogene
c-CBL is an adaptor protein that plays a crucial role in the
ubiquitylation of tyrosine kinase receptors such as epidermal growth
factor receptor and platelet-derived growth factor receptor (19, 21,
33, 34). c-CBL interacts with UbcH7 via its single RING-HC finger
domain (21), allowing c-CBL to function as an E3 ligase. The oncogenic 70Z-CBL isoform, which has a 17-amino acid deletion immediately upstream of the RING domain, fails to interact with UbcH7 (21) and, as
a consequence, fails to mediate ubiquitylation and degradation of
tyrosine kinase receptors (19, 21), which leads to their constitutive activation.
Other than the RING-HC finger domain c-CBL shares no other obvious
homologies in the domain or primary structure with HHARI and Parkin
(see Fig. 6A for a schematic representation of their protein
domain structures). The biological function of HHARI and its
orthologues remains unclear. By analogy with other proteins containing
RING domains that interact with UbcH7, it may possess an E3 activity.
In Drosophila, null mutants of Ariadne resulted in embryonic lethality at the pupal stage, with occasional adult survivors displaying severe disruption of the central nervous system
(14). This phenotype is analogous to mutants of the E2 encoded by
bendless (35). Indications of the function of HHARI will be
obtained with the identification of proteins that interact with it
in vivo and by mapping its interaction domains.
In this study, we demonstrate that HHARI binds and co-localizes with
UbcH7 in mammalian cells. We also identify the minimal interaction
region of HHARI (residues 186-254) and highlight key amino acid
residues within this domain that regulate the interaction. The distance
between the first RING finger and IBR domains is critical for UbcH7
binding. Moreover, we demonstrate that the RING finger motifs of c-CBL
and Parkin cannot substitute for that of HHARI to mediate binding of
the latter to UbcH7.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
HHARI coding sequence was
amplified by polymerase chain reaction with a primer to the vector T7
RNA polymerase promoter sequence and a reverse primer corresponding to
the 3'-end of HHARI using a full-length HHARI
cDNA clone isolated from the Origene fetal brain library (7). This
latter primer was designed to incorporate an EcoRI
restriction endonuclease site at its 5'-end. The amplification product
was digested with EcoRI, gel-purified, and cloned into pcDNA3.1(
)/Myc-His (Invitrogen). The stop codon of
HHARI was then altered from TGA to TGG using site-directed
mutagenesis to allow in-frame expression of the Myc-His tag.
UbcH7-GFP and UbcH8-GFP constructs were generated by
amplification of the inserts in previously described pET3a constructs (7), with primers incorporating either KpnI (UbcH7) or
BamHI (UbcH8) restriction endonuclease sequences in the
forward primer and an XhoI restriction endonuclease sequence
in the reverse primer. Amplification products were digested with the
appropriate restriction enzymes, gel-purified, and ligated into the
pcDNA3-derived GFP expression vector pJMA2eGFP (36).
All mutant HHARI constructs were generated using the QuikChange
site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. Deletion mutants were generated by
introducing restriction sites into mutant oligonucleotide primers
(either PmlI or HpaI; oligonucleotide sequences
available on request). One round of site-directed mutagenesis was
performed with one set of primers, and isolated clones were grown,
purified, and subjected to a second round of mutagenesis to generate
the second restriction site. Clones were then digested with the
appropriate restriction endonuclease(s), gel-purified, and religated to
create the deleted construct (details of individual constructs
available upon request).
HHARI-RINGcCBL and HHARI-RING1Parkin constructs were made using
HHARI-
RING1 as a template (Figs. 3A and 6B).
This latter construct retains only the first cysteine residue of the
RING1 finger domain. Polymerase chain reaction fragments for
HHARI-RINGcCBL encoding amino acid residues 383-420 of c-CBL or for
HHARI-RING1Parkin encoding residues 240-294 of Parkin were
generated by polymerase chain reaction using Pfu Turbo DNA
polymerase (Stratagene). These fragments were then inserted into
gel-purified, linear polymerase chain reaction product, which was
identical to that used to generate HHARI-
RING1.
Preparation of Antibody Solutions--
Mouse monoclonal anti-Myc
(clone 9E10) was obtained from Sigma. Living ColorsTM
anti-peptide antibody raised in rabbit for detection of GFP constructs was obtained from CLONTECH (herein abbreviated as
anti-GFP). The rabbit polyclonal anti-UbcH7 C-terminal peptide antibody
was described previously (7, 11). Polyclonal rabbit anti-HHARI
N-terminal peptide antibody (against MDSDEGYNYEFDED(C)) was generated
by Sigma Genosys. Antibody stock solutions were diluted 1:100 for anti-Myc and anti-GFP and diluted 1:5000 for anti-UbcH7 for Western blotting procedures. Secondary anti-rabbit and anti-mouse horseradish peroxidase-conjugated antibodies were purchased from Dako.
Anti-rabbit fluorescein isothiocyanate-conjugated and anti-mouse
tetra-methyl rhodamine isothiocyanate-conjugated
secondary antibodies were purchased from Vector Laboratories.
Cell Line Maintenance and Transfections--
Human kidney 293t
cells and monkey COS-7 cells were grown at 37 °C in 6%
CO2 in Dulbecco's minimal essential medium with Glutamax (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin. Transfections were performed in Dulbecco's minimal essential medium with
Glutamax supplemented with 2% (v/v) fetal calf serum using
LipofectAMINE (Life Technologies, Inc.) according to the
manufacturer's protocol.
Immunofluorescence and Confocal Microscopy for UbcH7 and
HHARI--
COS-7 cells were seeded onto sterile glass coverslips in a
6-well culture plate. Following attachment, the cells were transfected as described above with 2 µg of the appropriate construct(s). At
36 h post-transfection, coverslips were washed three times in
phosphate-buffered saline (PBS) followed by fixing with ice-cold methanol at
20 °C for 15 min. Subsequently, all procedures were performed at 20 °C. Post-fixation, coverslips were washed three times in PBS for 5 min prior to incubation with primary antibody solutions diluted 1:200 in 1% (w/v) nonfat milk in PBS for 2 h. Coverslips were then subjected to four 5-min washes with PBS before incubation with fluorescence-labeled secondary antibody solution at
1:100 dilution and 4,6-diamidino-2-phenylindole stain at 1:200 dilution in 1% (w/v) nonfat milk in PBS for 1 h. Following five 5-min washes in PBS, coverslips were mounted in Mowiol (Calbiochem) on
glass slides. Confocal images were obtained using a Leica TCS SP
confocal imaging system.
Immunoprecipitation of Proteins from Transfected Human Kidney
293t Cells--
Transfections were performed in 75-cm2
flasks using 6.6 µg of each construct DNA and 25 µl of
LipofectAMINE. 48 h post-transfection cells were washed twice in
PBS prior to the addition of 0.75 ml of prechilled lysis buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl,
1% (v/v) Nonidet P-40, 0.5% (v/v) sodium deoxycholate, and 1 × CompleteTM protease inhibitors (Roche Molecular
Biochemicals)) and incubation for 5 min at 20 °C. Cell suspensions
were homogenized using QIAshredder columns (Qiagen) followed by
centrifugation at 12,000 × g for 15 min at 4 °C.
Proteins were immunoprecipitated from supernatants using anti-Myc
antibody (diluted 1:100) and 50 µl (50%) of protein A-agarose
suspension according to the manufacturer's protocol (Roche Molecular
Biochemicals).
Western Blotting and Immunodetection--
Protein extracts were
resolved by SDS-polyacrylamide gel electrophoresis. Proteins
were then transferred on to Hybond-P polyvinylidene difluoride
membranes (Amersham Pharmacia Biotech). Nonspecific binding sites were
blocked in 3% (w/v) nonfat dried milk in PBS for 1 h at 20 °C
prior to incubation with primary antibody in 1% (w/v) nonfat dried
milk in PBS for 16 h at 20 °C. Bound primary antibody was
detected with horseradish peroxidase-conjugated secondary antibodies in
0.1% (w/v) nonfat milk in PBS. Enhanced chemiluminescence detection
was performed using SuperSignal® West Pico
chemiluminescent substrate (Pierce). When required, antibodies were
stripped from Western blots using 0.2 M sodium hydroxide
prior to reblocking with 3% (w/v) nonfat dried milk in PBS and
reprobing with additional antibody, as above.
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RESULTS |
HHARI Interacts with UbcH7 but Not with UbcH8 in Human Kidney 293t
Cells--
Co-transfection experiments with UbcH7-GFP and HHARI-Myc
constructs were performed to demonstrate interaction in
vivo. Both constructs produced readily detectable levels of
immunoreactive proteins (Fig. 1).
Expression of UbcH7 and/or UbcH7-GFP was demonstrated in cell lysates
with either affinity purified UbcH7 antibodies or anti-GFP antibodies
(Fig. 1A, lanes 7-12). Indeed, the presence of
endogenous UbcH7 observed in all lanes indicated approximately equal
loading of lysate. Immunoprecipitation of HHARI-Myc in cells transfected with HHARI-Myc or with HHARI-Myc and UbcH7-GFP led to the
co-precipitation of endogenous UbcH7 or both endogenous UbcH7 and
UbcH7-GFP, respectively (Fig. 1A, compare lanes 5 and 6). Immunoprecipitates of UbcH7-GFP were only observed
when this was co-transfected with HHARI-Myc (Fig. 1A,
compare upper and lower panels for lanes
4 and 6).

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Fig. 1.
HHARI interacts with UbcH7 but not UbcH8 in
293t cells. Myc-tagged HHARI in pcDNA3.1( )Myc/His together with
either GFP-tagged UbcH7 or control plasmids (pJMA2eGFP,
pcDNA3.1( )Myc/His) (A) and Myc-tagged HHARI in
pcDNA3.1( ) Myc/His together with either GFP-tagged UbcH7 or
GFP-tagged UbcH8 in pJMA2eGFP (B) were transfected into 293t
cells as described under "Experimental Procedures." 48 h
post-transfection tagged proteins were immunoprecipitated using
anti-Myc antibodies and size-fractionated by SDS-polyacrylamide gel
electrophoresis. Western blots of these gels were probed with either
affinity-purified anti-UbcH7 antibody (A, upper
panel) or anti-GFP antibodies (A, lower
panel, and B, upper panel) as indicated.
0.5% (v/v) of each of the total lysate volumes (nonimmunoprecipitated)
were run in parallel. The Western blot was stripped and reprobed with
either anti-GFP antibodies (A, upper panel) or
anti-Myc antibody (B, lower panel) to confirm
expression levels of transfected UbcH7-GFP or HHARI-Myc in 293t cells,
respectively. Molecular masses are indicated on the
left-hand column of each gel.
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No interaction was observed between HHARI-Myc and UbcH8 in
co-transfected 293t cells (Fig. 1B, lane 2). This
lack of interaction was not due to low levels of UbcH8 expression (Fig.
1B, lane 4). In this same experiment, HHARI-Myc
clearly immunoprecipitated UbcH7 (Fig. 1B, lane
1) when expressed at approximately the same levels as UbcH8 (Fig.
1B, compare lanes 3 and 4).
UbcH7 and HHARI Co-localize in COS-7 Cells--
We
performed immunofluorescence studies on COS-7 cells to investigate the
cellular localization patterns of UbcH7 and HHARI. Diffuse staining of
UbcH7 was observed within the nucleus and the cytoplasm of transfected
COS-7 cells, with extensive perinuclear staining (Fig.
2A). This staining pattern was
identical to that observed for the endogenous protein (data not shown).
HHARI was also detected within the cytoplasm with diffuse staining of
the nucleus (Fig. 2B). However, in cells overexpressing
HHARI the degree of nuclear staining was variable. Levels similar to
that of the endogenous protein were observed in some cells (Fig.
2B), whereas others displayed more intense nuclear staining
(Fig. 2C). Some perinuclear staining of HHARI was also
observed, although it was not as obvious as for UbcH7. Co-transfection
of UbcH7 and HHARI demonstrated areas of co-localization within cells,
primarily in the perinuclear region, although diffuse co-localization
was observed elsewhere (Fig. 2D, panel iv).
Co-staining with DAPI demonstrated that neither protein localized to
the nucleoli (Fig. 2, A-C, panels iii, and
D, panel iv).

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Fig. 2.
Co-localization of UbcH7 and HHARI in COS-7
cells. A, UbcH7-GFP was transfected into COS-7 cells.
36 h post-transfection, the cells were fixed in methanol and
immunostained with anti-GFP (panel i) and counterstained
with DAPI (panel ii) as described under "Experimental
Procedures." Panel iii represents an overlay of
panels i and ii. B and C,
COS-7 cells were transfected with HHARI-Myc and processed as in
A. Cells immunostained with anti-Myc (panel i)
and DAPI (panel ii). Panel iii represents an
overlay of panels i and ii. D, COS-7
cells transfected with UbcH7-GFP and HHARI-Myc and processed as in
A. The cells were immunostained with anti-GFP (panel
i) and anti-Myc (ii) and counterstained with DAPI
(panel iii). Panel iv represents the overlay of
panels i-iii. Yellow staining in these
overlays indicates co-localization of the two proteins within the COS-7
cells. Magnification, × 100.
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Establishing Which Domains of HHARI Regulate Its Interaction with
UbcH7 in Human Kidney 293t Cells--
Previous data from in
vitro experiments had indicated that the minimum region of HHARI
required for binding to UbcH7 was between amino acid residues 167 and
293. This region encompasses the first RING finger (RING1) and the
N-terminal part of the IBR domain. To confirm the interaction in
mammalian cells, each characterized domain was deleted from the
full-length HHARI-Myc construct as detailed in Fig.
3A, and the resulting deletion
constructs were co-transfected with UbcH7-GFP. Deletion of either
the acidic, the poly(Gly)-rich, or the second RING finger domain did
not diminish the interaction with UbcH7 (Fig. 3B,
upper panel, lanes 2, 3, and
6, respectively).Indeed, we consistently observed
increased interaction between HHARI-
Poly(Gly) and UbcH7, in repeat
experiments. We attribute this to increased levels of
HHARI-
Poly(Gly) after transfection, although we cannot rule out a
positive effect on the affinity of binding between UbcH7 and HHARI as a
result of deletion of the poly(Gly) sequence. By contrast, removal of
the first RING finger or the IBR domain prevented interaction with UbcH7 (Fig. 3B, upper panel, lanes 4 and 5, respectively). The absence of signal in the lanes
transfected with HHARI-
RING1 and HHARI-
IBR was not due to the
absence of mutant protein because reprobing the Western blot with the
anti-Myc antibody (Fig. 3B, lower panel) and the
presence of UbcH7-GFP in all of the cell lysates (Fig. 3B,
lanes 7-12) confirmed expression of these constructs.

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Fig. 3.
RING1 and IBR domains are required for the
interaction between UbcH7 and HHARI in 293t cells. A,
schematic illustrating the domain structure of HHARI (7) and the
deletion constructs produced in pcDNA3.1( )Myc/His. The
numbers above the lines indicate the positions of
amino acid residues. The spaces indicate where the peptide
sequences were deleted. In addition to the R-IBR-R motif (residues
186-375), HHARI contains an acidic region (22 of 30 acidic residues
between amino acids 11 and 41), a poly(Gly) region (20 of 25 residues
of Gly between amino acids 67 and 92; Ref. 7), and putative coiled-coil
and leucine zipper motifs within the C terminus of the protein (not
shown in this schematic; Ref. 14). B, co-transfections with
UbcH7-GFP into human kidney 293t cells and immunoprecipitation were
performed with each HHARI construct as described in the legend for Fig.
1. The interactions between proteins expressed from the HHARI-Myc
deletion constructs and UbcH7-GFP were first detected using
affinity-purified anti-UbcH7 antibodies (upper panel). The
blot was then stripped and reprobed with anti-Myc antibody to confirm
the expression levels of mutant HHARI in all lysates (lower
panel).
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Maintaining the Length of the "Spacer" Region between the RING1
Finger and IBR Domains Is Essential for the Interaction between UbcH7
and HHARI in Cells--
Deletion of the RING1 (HHARI-
RING1, deleted
residues 188-237) or the IBR (HHARI-
IBR, deleted residues 238-324)
domain prevented the interaction between UbcH7 and HHARI (Fig. 3).
Furthermore, our previous in vitro data indicated that a
minimal binding region, spanning amino acid residues 167-293, which
included the RING1 finger domain and part of the IBR domain, was
required for interaction. A construct consisting of HHARI residues
142-242 alone (RING1 with additional N- and C-terminal sequences) did
not bind UbcH7 (7, 11). We therefore postulated that a smaller
region of the sequence contained within the HHARI-
IBR construct must
be required for efficient binding of UbcH7.
A comparison of the primary structures of HHARI, H7-AP1, Parkin, and
other R-IBR-R-containing proteins, identified a common 20-amino acid
spacer region (amino acid residues 237-256 in HHARI) between the RING1
finger and IBR domains (Fig.
4A). These spacer regions
demonstrate no obvious sequence homology. The HHARI-
IBR construct
described above lacks both the IBR motif and this spacer region. A
series of Myc-tagged constructs were designed to test the hypothesis
that the presence and size of the spacer region may be important (Fig.
4B). HHARI-
238-254 was designed to delete the spacer
region; HHARI-
238-273 was designed to delete the spacer region and
part of the IBR but to leave the DRIL domain (residues 276-317)
intact; HHARI-
IBR(min) was designed to delete amino acid residues
254-324 of the IBR domain but leave the spacer region intact;
HHARI-
238-254HA was designed to delete residues 238-254, and a 9-amino acid HA tag was inserted to generate a 12-amino acid
spacer; and HHARI-237HA238 was designed to increase the size of the
spacer region to 29 amino acid residues by insertion of a 9-amino acid
HA tag between residues 237 and 238.

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Fig. 4.
Identification of an ESR between the RING1
finger and IBR domains of HHARI. A, alignment of the
ESRs between the RING1 finger and IBR domains of HHARI, Parkin, H7-AP1,
and TRIAD1 (alternatively termed hARI-2; Refs. 14 and 31). Amino acid
residue positions are indicated. B, schematic illustrating
the polypeptide sequence of HHARI encompassing the ESR and a
diagrammatic representation of the mutant constructs of HHARI generated
to investigate the role of the ESR (amino acid residues 237-256). The
C terminus of the RING1 finger and the N terminus of the IBR domain are
underlined. The various mutant proteins were as indicated.
The HA insertions consisted of the residues YPYDVPDYA. C,
co-transfections and immunoprecipitations of UbcH7-GFP with each HHARI
construct described for B were performed as described in the
legend for Fig. 1. Interacting proteins were immunoprecipitated with
anti-Myc antibodies, and UbcH7-GFP was detected using affinity-purified
anti-UbcH7 antibodies (upper panel). The blot was stripped
and reprobed with anti-Myc antibody to confirm expression levels from
mutant HHARI constructs in all lysates (lower panel).
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Expression of HHARI-
IBR(min) showed that it maintained its ability
to bind to UbcH7 (Fig. 4C, upper panel,
lane 6). In contrast, neither HHARI-
238-254 nor
HHARI-
238-273 was able to immunoprecipitate with UbcH7 (Fig.
4C, lanes 4 and 5, respectively).
Furthermore, replacement of the 20-amino acid spacer with a 12-amino
acid spacer or insertion of an additional sequence at the N terminus of
the spacer to create a 29-amino acid spacer abolished the UbcH7/HHARI interaction (Fig. 4C, lanes 2 and 3, respectively).
Identification of Amino Acid Residues in the RING1 Finger and IBR
Domains of HHARI That Regulate Interaction with UbcH7--
We next
introduced a series of deletions and mutations into the RING1 and IBR
domains of the HHARI-Myc construct (Fig.
5A). Single amino acid changes
or small deletions in Parkin and c-CBL are enough to prevent their
interactions with UbcH7, resulting in disease. We therefore focused
particularly on establishing whether equivalent residues in HHARI would
exert similar effects. The single and double mutants, HHARI-I188A and
HHARI-Q187H/I188V, were prepared as the equivalent Ile188
mutations in Parkin and c-CBL disrupt their interaction with UbcH7 (22,
37). Interestingly, although the single HHARI-I188A mutant lost its
ability to interact with UbcH7, the double mutant containing Q187H and
I188V did not (Fig. 5B, upper panel, compare lanes 3 and 4).

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Fig. 5.
Effects of single amino acid mutations in the
RING finger and IBR domains of HHARI on the interaction with
UbcH7. A, schematic illustrating the positions of the
point mutations and deletions of HHARI generated in
pcDNA3.1( )Myc/His as described under "Experimental
Procedures." Amino acid residues are as indicated. B,
co-transfections and immunoprecipitations of UbcH7-GFP with each HHARI
construct were performed as described in the legend for Fig. 1.
UbcH7-HHARI mutant interactions were detected using anti-GFP antibody
(upper panel) following immunoprecipitation with anti-Myc
antibodies. The blot was stripped and reprobed with anti-Myc antibody
to confirm approximately equal expression levels of the HHARI
constructs and loading of samples (lower panel).
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We noted that the RING finger domains of HHARI, Parkin, and c-CBL that
regulate their interactions with UbcH7 are all RING-HC types. To
establish whether RING proteins of the RING-H2 type might also be
likely to bind UbcH7, we changed Cys208 to
His208 at the variable Cys/His5 position of the
RING1 domain of HHARI-Myc to create the construct HHARI-C208H. Its
activity was compared with both HHARI-Myc and with HHARI-C208A.
Alteration of Cys208 to either His208 or
Ala208 abolished the interaction of HHARI with UbcH7 (Fig.
5B, upper panel, compare lanes 5 and
6 with lane 1). The absence of co-precipitation was not due to the lack of expression of the construct or the instability of the expressed protein, as evidenced by the approximately equal levels of Myc-tagged proteins (Fig. 5B, lower
panel, lanes 1-6). On comparing proteins of the
R-IBR-R family including Parkin, Tyr258 is conserved,
implying that it may be structurally significant for binding UbcH7
(30). We therefore generated and tested HHARI-Y258A for its ability to
immunoprecipitate with UbcH7 in cells. This mutant retained the ability
to interact with UbcH7 (Fig. 5B, upper panel,
lane 7).
Generation of the HHARI-
163-187 construct addresses two questions.
First, removal of amino acids 163-187 creates a HHARI mutant
equivalent to the 70Z oncogenic mutant of c-CBL. This latter protein
prevents UbcH7 binding and subsequent c-CBL ubiquitylation (21).
Second, the construct clarifies whether HHARI requires any sequence
N-terminal to the RING1 finger sequence for its interaction with UbcH7.
Although this construct lacked the first cysteine residue,
Cys186, of the RING1 finger motif, the generation of
HHARI-
163-187 (Fig. 5A) brought Cys161 into
an equivalent position while at the same time deleting the intervening
sequence. This construct retained full UbcH7 binding activity (Fig.
5B, upper panel, lane 2).
RING Finger Domains of Parkin or c-CBL Cannot Substitute for the
RING1 Finger Domain of HHARI--
The interactions of HHARI, Parkin,
and c-CBL with UbcH7 are all regulated through their RING-HC finger
domains. This suggested that these domains might be interchangeable
between the three proteins. To test this hypothesis, RING1 of Parkin or
the RING domain of c-CBL was incorporated into the HHARI-
RING1
template (Figs. 3A and
6B). Both HHARI-RING1Parkin
and HHARI-RINGcCBL retain the 20-amino acid spacer of HHARI. The
heterologous RING finger fusion proteins proved incapable of
interacting with UbcH7-GFP (Fig. 6C, upper panel,
lanes 2 and 3) despite being expressed at levels
approximately equal to that of HHARI-Myc (Fig. 6C,
lower panel, lanes 1-3). These data suggest a
higher order of complexity in the interactions between UbcH7 and other
proteins over and above that provided by RING domains alone.

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Fig. 6.
RING finger domains of Parkin or c-CBL
cannot substitute for the RING1 finger domain of HHARI.
A, schematic illustrating the domain structures of HHARI,
Parkin, and c-CBL. All three proteins share a RING-HC finger domain.
HHARI and Parkin also contain the ESR, the IBR, and an additional
RING-HC finger domain. B, a comparison of the structure of
the first RING finger domain of HHARI with that of Parkin and with the
RING domain of c-CBL. Amino acid residue positions are as indicated.
The cysteine and histidine residues that define the RING finger domains
are underlined. A dash denotes a shift in amino
acid sequence to maintain alignment. C, HHARI-RINGcCBL
and HHARI-RING1Parkin constructs were prepared in
pcDNA3.1( )Myc/His as described under "Experimental
Procedures." These constructs were co-transfected with UbcH7-GFP into
human kidney 293t cells as described in the legend for Fig. 1. The
interactions between UbcH7-GFP and heterologous HHARI fusion proteins
were detected using affinity-purified anti-UbcH7 antibodies
(upper panel) following immunoprecipitation with anti-Myc
antibodies. The blot was stripped and reprobed with anti-Myc antibody
to confirm equivalent expression levels of mutant HHARIs in all cell
lysates and immunoprecipitates (lower panel).
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DISCUSSION |
We have mapped in detail the RING1 finger-IBR region of HHARI
required for binding to UbcH7 in cells. This sequence (residues 186-254), which significantly refines that demonstrated in our previous in vitro studies (residues 167-293), spans the
first RING domain and an essential spacer region (ESR) of 20 amino
acids between the first RING and the IBR domain. Although the ESR is found in the majority of other R-IBR-R proteins, including H7-AP1, Parkin, and TRIAD1/hAri-2 (a protein closely related to HHARI that can
also interact with UbcH7 (14, 31), there is little sequence homology
within the ESR. Its size appears to be important, because spacers
consisting of 3, 12, or 29 amino acid residues (constructs
HHARI-
238-254, HHARI-
238-254HA, and HHARI-237HA238, respectively) could no longer support the interaction between UbcH7 and
HHARI. Hence, we speculate that its function is to keep the RING1 and
IBR domains precisely positioned to permit specific folding and interactions.
In vitro studies had indicated that HHARI was capable of
binding to UbcH7 and the closely related protein UbcH8. However, experiments herein indicate that HHARI interacts only with UbcH7 in
cells. The reason for this difference is not clear. It may reflect
competition between endogenous UbcH7 and transfected UbcH8 for binding
to HHARI, if the affinity for UbcH7 is greater than that for UbcH8. In
this regard, Shimura et al. (22) demonstrated that 36 times
the levels of UbcH8 compared with UbcH7 were required to bind
approximately equivalent amounts of Parkin. Furthermore, Martinez-Noel
et al. (12) identified a family of proteins containing RING
finger domains that bound to the UbcH7 murine orthologue, UbcM4, but
not to other E2s, including UbcH8. It is unlikely that the observed
lack of interaction between UbcH8 and HHARI is due to structural
constraints imposed by the presence of the GFP tag, because
C-terminally tagged UbcH8 was capable of interacting with HHARI in
noncompetitive in vitro assays (7). Moreover, there is a
very high degree of similarity in size and structure between UbcH7 and
UbcH8. These data indicate that an interaction of UbcH8 with either
HHARI or Parkin probably does not usually occur under normal
physiological conditions.
Subtleties in the interaction between HHARI and UbcH7 were further
suggested by the observation that mutation of Cys208 to
His208, which effectively changes the RING1 finger domain
of HHARI from a RING-HC type to a RING-H2 type, abolished its ability
to bind UbcH7. This is despite previous observations that other E2s,
such as UbcH5, can interact with proteins containing either RING-H2 or
RING-HC type domains (20). Furthermore, mutations of single amino acid
residues in HHARI, equivalent to those found in Parkin or c-CBL that
led to loss of UbcH7 binding, had a similar effect on HHARI. The most
interesting were those involving residue Ile188. The double
mutant HHARI-Q187H/I188V bound UbcH7, whereas HHARI-I188A alone did
not. Clues to the cause of this phenomenon are provided by the recently
published c-CBL/UbcH7 crystal structure (37). The equivalent residue in
c-CBL at Ile383 is required for interaction with the
Phe63 residue of the highly conserved central domain found
within UbcH7 and other E2s that lies immediately N-terminal to the
active site central Cys86 residue. Moreover, an autosomal
recessive juvenile Parkinsonism-associated mutation in Parkin, T240R
(38), at the site corresponding to Ile188, prevented
binding with UbcH7 and subsequent E3 ligase activity of the protein
(22).
Removal of the acidic, poly(Gly), or RING2 finger domains had little
effect on HHARI-UbcH7 binding. However, it is interesting to note that
elevated levels of binding between HHARI-
Poly(Gly) and UbcH7-GFP
were consistently observed in repeat experiments, regardless of the
amount of DNA transfected into the cells. Several recent studies have
indicated that poly(Gly) or poly(Gly-Ala) motifs may serve as
inhibitors of proteasome-mediated degradation (39, 40).
The cellular distributions of different RING finger proteins reflect
the diverse roles played by these proteins in cells. Some such as PML
and TRIAD1 are exclusively nuclear (41-43), whereas others are mainly
cytoplasmic (44, 45). HHARI appears to localize mostly to the
cytoplasm, although some cells demonstrated intense nuclear staining
after transfection. Although this may somehow relate to high levels of
HHARI expression, it is difficult to explain why this should result in
the translocation of such a large protein into the nucleus. It may
reflect a change in the proliferation/differentiation status of the
cell. Our subcellular localization studies of both endogenous UbcH7 and
transfected UbcH7-GFP confirmed the results of Anan et al.
(46). Co-expression of both UbcH7 and HHARI demonstrated that a high
proportion of both proteins co-localized in the cytoplasm, particularly
at the nuclear periphery. At this stage we have no explanation as to why UbcH7 and HHARI should demonstrate perinuclear co-localization, although intriguingly this is also the site of proteasomal subunit accumulation (47, 48).
We had hypothesized that because the UbcH7-interacting RING finger
domain structures of HHARI, Parkin, and c-CBL are so similar, they
might be functionally interchangeable (21, 22). To test this concept,
we generated heterologous HHARI proteins in which the RING1 finger
domain was replaced with either the RING1 finger of Parkin or the RING
finger polypeptide sequence of c-CBL. However, absence of binding was
observed with both of these heterologous proteins. These data suggested
that at the three-dimensional structure level small differences in
amino acid sequences between the RING fingers cannot be compensated for
by other sequences outside this domain. Indeed, Parkin has been
demonstrated to require the IBR and the second RING finger domain (22),
whereas c-CBL requires sequences N-terminal to the RING domain (21) for
binding to UbcH7. It is interesting that in contrast to the data
presented herein, a heterologous MDM2 protein containing the Praja1
RING domain was able to interact with UbcH5B and drive
E2-dependent ubiquitylation/degradation of itself but could
not support ubiquitin modification of its target, p53 (5). Thus, much
remains to be elucidated before we fully understand the complex
interplay of E2s and their RING finger protein partners.