Features of the Parkin/Ariadne-like Ubiquitin Ligase, HHARI, That Regulate Its Interaction with the Ubiquitin-conjugating Enzyme, UbcH7*

Helen C. ArdleyDagger §, Nancy G. S. TanDagger , Stephen A. RoseDagger , Alexander F. MarkhamDagger , and Philip A. RobinsonDagger

From the Dagger  Molecular Medicine Unit and the  Leeds Dental Institute, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, United Kingdom

Received for publication, December 7, 2000, and in revised form, February 21, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We recently reported the identification of a RING finger-containing protein, HHARI (human homologue of Drosophila ariadne), which binds to the human ubiquitin-conjugating enzyme UbcH7 in vitro. We now demonstrate that HHARI interacts and co-localizes with UbcH7 in mammalian cells, particularly in the perinuclear region. We have further defined a minimal interaction region of HHARI comprising residues 186-254, identified individual amino acid residues essential for the interaction, and determined that the distance between the RING1 finger and IBR (in between RING fingers) domains is critical to maintaining binding. We have also established that the RING1 finger of HHARI cannot be substituted for by the highly homologous RING finger domains of either of the ubiquitin-protein ligase components c-CBL or Parkin, despite their similarity in structure and their independent capabilities to bind UbcH7. Furthermore, mutation of the RING1 finger domain of HHARI from a RING-HC to a RING-H2 type abolishes interaction with UbcH7. These studies demonstrate that very subtle changes to the domains that regulate recognition between highly conserved components of the ubiquitin pathway can dramatically affect their ability to interact.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 epsilon -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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-Delta 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-Delta 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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-Delta Poly(Gly) and UbcH7, in repeat experiments. We attribute this to increased levels of HHARI-Delta 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-Delta RING1 and HHARI-Delta 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).

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-Delta RING1, deleted residues 188-237) or the IBR (HHARI-Delta 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-Delta 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-Delta 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-Delta 238-254 was designed to delete the spacer region; HHARI-Delta 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-Delta IBR(min) was designed to delete amino acid residues 254-324 of the IBR domain but leave the spacer region intact; HHARI-Delta 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).

Expression of HHARI-Delta IBR(min) showed that it maintained its ability to bind to UbcH7 (Fig. 4C, upper panel, lane 6). In contrast, neither HHARI-Delta 238-254 nor HHARI-Delta 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).

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-Delta 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-Delta 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-Delta 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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-Delta 238-254, HHARI-Delta 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-Delta 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.

    ACKNOWLEDGEMENTS

We thank Dr. J. M. Askham for the pJMA2eGFP construct and Dr. E. E. Morrison for useful discussions concerning confocal imaging.

    FOOTNOTES

* This work was supported by the Wellcome Trust, the Royal Society, Yorkshire Cancer Research, and a Lloyds of London Tercentenary Foundation Fellowship (to H. C. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Molecular Medicine Unit, Level 6, Clinical Sciences Bldg., St. James's University Hospital, Leeds LS9 7TF, UK. Tel.: 44-113-2065682; Fax: 44-113-2444475; E-mail: rmrhca@stjames.leeds.ac.uk.

Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M011028200

    ABBREVIATIONS

The abbreviations used are: E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; ESR, essential spacer region; R-IBR-R, RING-IBR-RING; GFP, green fluorescent protein; HA, human influenza virus hemagglutinin; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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