c-MIR, a Human E3 Ubiquitin Ligase, Is a Functional Homolog of Herpesvirus Proteins MIR1 and MIR2 and Has Similar Activity*

Eiji GotoDagger, Satoshi IshidoDagger§, Yuya Sato, Shinji Ohgimoto, Kaori Ohgimoto, Motoko Nagano-Fujii, and Hak Hotta

From the Division of Microbiology, Department of Genome Sciences, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan

Received for publication, November 5, 2002, and in revised form, January 17, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Kaposi's sarcoma associated-herpes virus encodes two proteins, MIR (modulator of immune recognition) 1 and 2, which are involved in the evasion of host immunity. MIR1 and 2 have been shown to function as an E3 ubiquitin ligase for immune recognition-related molecules (e.g. major histocompatibility complex class I, B7-2, and ICAM-1) through the BKS (bovine herpesvirus 4, Kaposi's sarcoma associated-herpes virus, and Swinepox virus) subclass of plant homeodomain (PHD) domain, termed the BKS-PHD domain. Here we show that the human genome also encodes a novel BKS-PHD domain-containing protein that functions as an E3 ubiquitin ligase and whose putative substrate is the B7-2 co-stimulatory molecule. This novel E3 ubiquitin ligase was designated as c-MIR (cellular MIR) based on its functional and structural similarity to MIR1 and 2. Forced expression of c-MIR induced specific down-regulation of B7-2 surface expression through ubiquitination, rapid endocytosis, and lysosomal degradation of the target molecule. This specific targeting was dependent upon the binding of c-MIR to B7-2. Replacing the BKS-PHD domain of MIR1 with the corresponding domain of c-MIR did not alter MIR1 function. The discovery of c-MIR, a novel E3 ubiquitin ligase, highlights the possibility that viral immune regulatory proteins originated in the host genome and presents unique functions of BKS-PHD domain-containing proteins in mammals.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previously, we and other groups found that Kaposi's sarcoma associated-herpesvirus (KSHV)1 MIR (modulator of immune recognition), MIR2 proteins, and murine gamma -herpesvirus 68 K3 protein down-regulate the surface expression of MHC class I (MHC I) on several cell lines (1-4). Recently, these viral proteins have been shown to ubiquitinate MHC I (5-7). Notably, MIR1 and MIR2 have been shown to function as E3 ubiquitin ligase and the BKS (bovine herpesvirus 4, KSHV, and Swinepox virus) subclass of plant homeodomain (PHD) domain, termed the BKS-PHD domain, was identified as a catalytic domain of their E3 ubiquitin ligase activity (6). The BKS-PHD domain was designated based on its existence in bovine herpesvirus, KSHV, and Swinepox virus genome (8). This domain was classified as a subclass of the PHD domain because of an interval difference between the third and fourth cysteine residues in a zinc-binding motif. The PHD domain has been found in many proteins involved in chromatin-mediated transcriptional regulation, but their functions remain unknown. Although KSHV MIRs and murine gamma -herpesvirus 68 K3 share the same structure and the same positioning of BKS-PHD, they utilize distinct pathways for degradation of MHC I; KSHV MIRs lead target molecules to lysosomal degradation (1, 9), whereas murine gamma -herpesvirus 68 K3 causes proteasomal degradation (3, 5). Nevertheless, the BKS-PHD domain has been shown to be a critical domain for ubiquitination and degradation of MHC I in all cases.

The PHD domain has a similar structure to the RING (really interesting new gene) domain, which is a functionally critical domain for several E3 ubiquitin ligases such as c-Cbl and Hakai (10-12). c-Cbl and Hakai induce the ubiquitination of receptor or nonreceptor tyrosine kinase and E-cadherin, respectively (10, 12, 13). c-Cbl and Hakai-mediated ubiquitination requires tyrosine phosphorylation of targets and induces rapid endocytosis and lysosomal degradation of targets (10, 12, 13). This is the case with MIR1 and MIR2. The expression of MIR1 or MIR2 induces rapid endocytosis, translocation to the trans-Golgi network, and lysosomal degradation of MHC I (1, 9). The BKS-PHD domain of MIR1 has been shown to be a functionally critical domain for the endocytosis of MHC I (9). Recently, the PHD domain of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 has also been shown to function as an E3 ubiquitin ligase and to induce ERK degradation in a proteasome-dependent manner (14).

These findings gave rise to the question as to where the viral BKS-PHD domain-containing proteins are derived from. The genomes of large DNA viruses including herpesviruses carry many homologs of host cellular proteins. Likewise, the KSHV genome encodes homologs of many human genes (e.g. viral interleukin-6 (v-IL-6) and viral macrophage inflammatory protein (v-MIP)), which have been implicated in viral pathogenesis (15), suggesting that viral BKS-PHD domain-containing proteins might also have originated in the host genome. To justify this hypothesis, we searched the human protein data base of Celera Genomics (16). Bioinformatics and experimental analysis led us to discover that the human genome contains a gene encoding a novel BKS-PHD domain-containing protein that functions as an E3 ubiquitin ligase and whose putative substrate is the B7-2 co-stimulatory molecule. Based on its functional and structural similarity to KSHV MIR1 and MIR2, we have designated this novel E3 ubiquitin ligase as c-MIR (cellular MIR). Although overall sequence similarity between c-MIR and KSHV MIRs is low, they share the same secondary structure. Forced expression of c-MIR causes polyubiquitination, rapid endocytosis, and lysosomal degradation of B7-2 specifically. This specific targeting is achieved through binding to the transmembrane and/or cytoplasmic regions of B7-2. Furthermore, the BKS-PHD domain of c-MIR functioned in the context of KSHV MIR1. This finding highlights the possibility that viral immune regulatory proteins originated in the host genome and shows unique functions of mammalian BKS-PHD domain-containing proteins.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Data Base Searches and Cloning cDNA Encoding Human c-MIR-- To look for the candidates of KSHV MIR1,2 homolog, the data base of Celera Genomics was searched using the program BLAST-P, which allowed a search for patterns in translated nucleic acid sequences data bases (16). For cloning of the cDNA coding human c-MIR, total RNA isolated from BJAB cells were reverse-transcribed using the SuperScript RT kit (Invitrogen) according to the manufacturer's protocol. To determine the 5' and 3' ends of the entire coding sequence of c-MIR cDNA, 5' and 3' rapid amplification of cDNA ends were performed using a GeneRacerTM kit (Invitrogen). A full-length cDNA of c-MIR was obtained by PCR and subcloned into the pEF-1 vector and GFP co-expression vector pTracer EF-1 (Invitrogen) and sequenced by a model 310 DNA sequencer (Applied Biosystems). The putative secondary structure and transmembrane topology of c-MIR were examined by Profile fed neural network system from HeiDelberg (PHD) (17-19).

Detection of c-MIR mRNA from Human Tissues and Cultured Cells-- For the detection of c-MIR mRNA from human tissues, PCR-ready cDNA kits (Maxim Biotech) were subjected to PCR analysis with the specific primers, 5'-CGCGAATTCGCCGCCATGAGCATGCCACTG-3' (forward) and 5'-CGCTCTAGAGACGTGAATGATTTCTGCTCC-3' (reverse) to detect the full-length human c-MIR mRNA according to the manufacturer's protocol. For detection of mRNA from cultured cells, total RNA was extracted by using the RNeasy mini kit (Qiagen), and 2 µg of total RNA was reverse-transcribed. 400 ng of cDNA was subjected to PCR analysis as described above. Each PCR product was subcloned into pEF-1 and verified by DNA sequencing.

Generation of Antibody-- Antibody directed against c-MIR was produced by immunizing rabbits with a synthetic peptide, DAISARVYRSKTKEKEREE, corresponding to amino acids of 15-33 of c-MIR. The rabbits were immunized with keyhole limpet hemocyanin-coupled peptides in complete adjuvant, followed by antisera preparation.

Quantification by Real Time RT-PCR-- A one-step RT-PCR analysis was performed using the QuantiTectTM SYBR Green RT-PCR kit (Qiagen). 50 ng of total RNA was analyzed according to the manufacturer's recommendation. The SYBR Green fluorescence was measured after each elongation using an ABI PRISM 7000 sequence detection system (Applied Biosystems). After PCR amplification, a melting curve analysis was performed by increasing the temperature from 60 to 95 °C. To assess the purity of the amplicons of interest, RT-PCR products were analyzed by gel electrophoresis. The intensity of the SYBR Green fluorescent signal was converted into a relative number of copies of interest based on the results of a series of standards prepared by successive dilution of total RNA. The expression level of glyceraldehye-3-phosphate dehydrogenase (GAPDH) was determined for normalization of the data set. Each experiment was performed twice using duplicate samples from independently generated cDNA templates.

Plasmid Construction-- B7-2 cDNA was obtained from total RNA of BJAB cells by RT-PCR and subcloned into pEF-1. HLA-A2 and CD8 cDNA were kindly provided by Dr. G. Cohen (20). Each CD8 chimera was constructed by overlapping PCR as described previously (2, 21). Substitutions were engineered into each chimera by PCR-based mutagenesis (Promega). For analysis by two-color flow cytometry, each cDNA was subcloned into the GFP co-expression vector pTracer EF-1. To introduce a FLAG epitope tag, each cDNA was subcloned into p3XFLAG-CMV vector (Sigma). To construct plasmid DNAs for GST fusion proteins, a fragment encoding the c-MIR BKS-PHD domain was amplified by PCR and then subcloned into pGEX 4T-1 (Amersham Biosciences).

Cell Culture and Transfection-- BJAB, 293T, and A7 cells (ATCC) were grown in RPMI with 10% fetal calf serum or Dulbecco's modified Eagle's medium with 10% fetal calf serum and minimum essential medium with 10% fetal calf serum. For transient assays, expression plasmid DNAs were introduced by electroporation at 260 V and 975 microfarads in serum-free RPMI medium or by transfection with FuGENE 6 reagent (Roche Molecular Biochemicals). To select stable transformants, 2 mg/ml of G418 (Sigma) was added 48 h post-transfection, and the cells were maintained in selective medium for 3-6 weeks.

Monocytes and Dendritic Cells (DCs)-- Human peripheral blood mononuclear cells (PBMCs) from healthy volunteers were separated from peripheral blood by Ficoll-Hypaque centrifugation. The monocytes were obtained from PBMCs using the MACS monocyte isolation kit (Miltenyi Biotec), which depletes T cells, NK cells, B cells, and basophils from PBMC using a mixture of hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, and anti-IgE antibodies. Immature DCs were generated by culturing monocytes at a concentration of 1 × 106 cells/ml for 7 days; granulocyte-macrophage colony-stimulating factor (2000 units/ml; R&D Systems) and interleukin-4 (3000 units/ml; R&D Systems) were added on days 0 and 4. The cell fraction remaining after monocyte isolation was collected and used as a source of peripheral blood lymphocytes (PBLs).

Metabolic Labeling, Immunoprecipitation, and PNGase-F Digestion-- For metabolic labeling, the cells were washed three times with phosphate-buffered saline (PBS), washed once with labeling medium (RPMI minus methionine and cysteine plus 10% dialyzed fetal calf serum), and then incubated with 5 ml of the same medium containing 50 µCi of [35S]methionine and [35S]cysteine (PerkinElmer Life Sciences) for 6 h. For pulse-chase analysis, the cells were labeled for 30 min and chased for the indicated time. For immunoprecipitation, the cells were harvested and lysed with lysis buffer (0.15 M NaCl, 1% Nonidet P-40, and 50 mM HEPES buffer, pH 8.0) containing protease inhibitors. Immunoprecipitation was performed with the indicated antibody together with 30 µl of protein A/G-agarose beads (Santa Cruz). For PNGase-F digestion, washed immunoprecipitates were resuspended in 50 µl of 1× denaturing buffer (0.5% SDS, 1% beta -mercaptoethanol), heated for 10 min at 100 °C, and incubated for 1 h at 37 °C with 1 µl of PNGase-F (New England Biolabs).

Immunofluorescence Microscopy-- The cells were fixed with 4% paraformaldehyde PBS for 15 min and cold acetone for 15 min. Fixed cells were stained with 1:100 diluted primary antibody in PBS for 30 min. After incubation, the cells were washed extensively with PBS and incubated with a 1:1000 dilution of Alexa 488 or 568-conjugated secondary antibody (Molecular Probes) in PBS for 30 min. Finally, the cells were washed three times with PBS and mounted in mounting medium (Vector).

Flow Cytometry Analysis and Antibodies-- The cells (5 × 105) were washed with RPMI medium containing 2% fetal calf serum and incubated with fluorescein isothiocyanate- or phycoerythrin (PE)-conjugated monoclonal antibodies for 30 min at 4 °C. After being washed, each sample was fixed with 2% paraformaldehyde solution, and flow cytometry analysis was performed with a FACScan (Becton Dickinson). W6/32 antibody for MHC I, RPA-T8 antibody for CD8, HA 58 antibody for ICAM-1, L307.4 for B7-1, and FUN-1 antibody for B7-2 used for FACScan were obtained from PharMingen Becton Dickinson Company. The M2 anti-FLAG antibody (Sigma), F7 anti-HA antibody (Santa Cruz), G-18 anti-His antibody (Santa Cruz), P4D1 anti-ubiquitin antibody (Santa Cruz), and the anti-V5 antibody (Invitrogen) were used for immunoprecipitation and/or immunoblot analysis.

Endocytosis Assay-- The experiments were performed as described previously (2, 9). Briefly, c-MIR cells or control BJAB cells were stained with PE-labeled anti-B7-2 antibody at 4 °C and then incubated for various periods of time at 37 °C. The cells were then washed in an acidic solution to remove uninternalized antibodies, fixed, and subjected to flow cytometry. For confocal microscopy analysis, fluorescein isothiocyanate-labeled B7-2 antibody was added to the culture medium of control BJAB cells or c-MIR cells and then incubated for 2 h at 37 °C. After being washed, these cells were subjected to analysis with a confocal immunofluorescence microscope (Bio-Rad).

Immunoprecipitation and Immunoblots-- Each plasmid DNA was transfected into 293T cells using the FuGENE 6 reagent (Roche Molecular Biochemicals). After 48 h, the transfected cells were harvested, lysed with Nonidet P-40 buffer, immunoprecipitated as described above, and subjected to serial immunoblots as indicated in each experiment.

Ubiquitination Assay-- For in vitro auto-ubiquitination assay, the GST fusion proteins were produced as follow. Expression of GST fusion proteins was induced by 0.1 mM isopropyl-1-beta -D-thio-galactopyranoside for 3-6 h. Bacterial pellets were sonicated in PBS containing protease inhibitors and 0.1% Triton X-100. After being cleared by centrifugation, bacterial lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences). 10 µg of precipitated GST fusion proteins were mixed with 1 µg of UbcH5a (Boston Biochem), 2 µg of His-tagged ubiquitin (Calbiochem), and 40 ng of E1 (Calbiochem) in the auto-ubiquitination buffer (40 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM ATP, 2 mM dithiothreitol, 25 µM MG132), incubated at 35 °C for 3 h, and subjected to immunoblot with anti-ubiquitin antibody.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of a Functional Homolog of KSHV MIR1 and MIR2 in the Human Genome-- We found a BKS-PHD domain-containing putative protein, hCP36279, that shares the same secondary structure and the same positioning of BKS-PHD domain with MIR1 and MIR2 in the data base of Celera Genomics (16). Because amino-terminal sequences of hCP36279 were missing in the public data base, 5' rapid amplification of cDNA ends analysis was performed using cap-trapping methods. We determined the full sequence of an hCP36279 cDNA cloned from the BJAB B cell line (Fig. 1A). Overall hCP36279 has 12 and 18% amino acid identity to MIR1 and MIR2, respectively. However, within their BKS-PHD domains, hCP36279 exhibits 36 and 42% amino acid identity compared with MIR1 and MIR2, respectively (Fig. 1B). hCP36279 was predicted to have two intracytoplasmic regions, helical transmembrane regions separated by an extra-cytoplasmic domain, and a BKS-PHD domain located in the putative amino-terminal intracytoplasmic region. The predicted structure of hCP36279 is depicted schematically, together with those of MIR1 and MIR2, in Fig. 1C. To examine the functional similarity of this protein to MIR1 and MIR2, His-tagged protein was transiently expressed in BJAB cells by electroporation, and the surface expression of immune recognition-related molecules was analyzed by two-color flow cytometry. As shown in Fig. 2A, B7-2 surface expression was significantly down-regulated, but not MHC I and ICAM-1. The FLAG-tagged version also exhibited the same function (data not shown). Based on these findings, this protein has been designated as c-MIR. To confirm these findings, BJAB cell lines were engineered to have stable and excess expression of c-MIR (c-MIR cells) and examined for surface expression of immune recognition-related molecules by flow cytometry. In this experiment, the examination of B7-1 was included to confirm the target specificity of c-MIR. The stable expression of exogenous c-MIR (His-tagged c-MIR) was confirmed by immunoprecipitation from the metabolically labeled cell lysate with an anti-His antibody (Fig. 2B, left panel). Real time RT-PCR analysis showed that the c-MIR expression level of c-MIR cells was 57-fold higher than that of control BJAB cells (Fig. 2B, right panel). Flow cytometry analysis showed that the surface expression of B7-2 was specifically down-regulated on c-MIR cells (Fig. 2C).


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Fig. 1.   hCP36279 is a structural homolog of KSHV MIR1 and MIR2. A, the amino acid sequence of hCP36279 is shown. Two transmembrane domains (shown as TM1 and TM2) were predicted by the PHDhtm program. Underlining indicates consensus residues of the BKS-PHD domain. B, the alignment of protein sequences from hCP36279, MIR1, and MIR2 within their BKS-PHD domains. The putative zinc-binding residues are marked by asterisks. C, a putative molecular structure and transmembrane topology of hCP36279 are shown together with MIR1 and MIR2. The BKS-PHD domain is shown as PHD. The putative secondary structure and transmembrane topology of c-MIR were determined by using Profile fed neural network system from HeiDelberg (17-19).


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Fig. 2.   hCP36279, renamed as c-MIR, is a functional homolog of KSHV MIR1 and MIR2. A, BJAB cells were transfected with the GFP-c-MIR vector by electroporation. The surface expression of each molecule and the expression of GFP were analyzed 24 h post-transfection by two-color flow cytometry as in previous experiments (2, 9). In brief, the cells (5 × 105) were washed with RPMI medium containing 2% fetal calf serum and incubated with the indicated PE-conjugated monoclonal antibodies for 30 min at 4 °C. After being washed, each sample was fixed with 2% paraformaldehyde solution, and flow cytometry analysis was performed with a FACScan (Becton Dickinson). The y axis shows the level of surface expression of MHC I, ICAM-1, and B7-2, and the x axis shows the level of GFP expression. B, the left panel shows the stable expression of exogenous c-MIR protein in c-MIR cells. c-MIR cells and control BJAB cells were labeled with [35S]methionine and [35S]cysteine for 6 h, and metabolically labeled cell lysates were immunoprecipitated with anti-His polyclonal antibody. Immunoprecipitates were analyzed by SDS-PAGE. In the right panel, the level of c-MIR mRNA was determined by using quantitative RT-PCR in control BJAB cells and c-MIR cells. The expression level of c-MIR was normalized to GAPDH expression and is indicated relative to the expression level in control BJAB cells. All of the reactions were performed in duplicate, and the standard deviation is indicated. C, BJAB cells stably overexpressing c-MIR (c-MIR) and control BJAB cells were stained with the indicated antibodies as described for A, and the surface expression of the indicated molecules was analyzed by flow cytometry. In merged panels, the bold lines and shaded histograms show the results of c-MIR cells and control BJAB cells, respectively. The results of the staining with isotype control antibody are shown as dotted lines.

Detection of c-MIR mRNA and Protein in Human Monocyte-derived DCs-- As shown in Fig. 2, c-MIR is able to down-regulate the surface expression of B7-2 in BJAB cells. This finding suggests that c-MIR might be a functional molecule in antigen-presenting cells, because B7-2 is a co-stimulatory molecule for antigen presentation. To test this hypothesis, the expression profile of c-MIR mRNA in various tissues was examined by RT-PCR. The primer set used in this experiment is able to detect full-length c-MIR mRNA (873 bp). As shown in Fig. 3A, full-length c-MIR mRNA was detected in neonatal brain, lymph node, spleen, and placenta. The nucleotide sequence of all PCR products was exactly the same as that of cDNA derived from BJAB cells. In addition, a band slightly bigger than 873 bp was detected in neonatal brain. DNA sequencing analysis revealed that this transcript had additional sequences within full-length c-MIR mRNA, suggesting that it is an immature transcript of c-MIR.


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Fig. 3.   Expression of c-MIR. A, 20 ng of cDNA from neonatal brain, lymph node, heart, lung, liver, spleen, kidney, and placenta were PCR-amplified using the primers described under "Experimental Procedures" to detect full-length c-MIR mRNA (upper panel). Also the expression of GAPDH mRNA was measured (lower panel). B, DCs, monocytes and PBLs were prepared from human PBMCs as described under "Experimental Procedures." Total RNA from these cells was reverse-transcribed and PCR-amplified as performed in A (upper panel). The lower panel shows the expression of GAPDH mRNA. C, DCs, c-MIR-expressed 293T (293T+c-MIR), and 293T were labeled with [35S]methionine and [35S]cysteine for 6 h. Each labeled cell extract was immunoprecipitated with anti-c-MIR polyclonal antibody (alpha -c-MIR) or preimmune serum (Cont). Each precipitated protein sample was analyzed by SDS-PAGE. The bands corresponding to the authentic c-MIR protein are marked with asterisks.

Because full-length c-MIR mRNA was detected in lymphoid tissues, we sought to determine whether DCs, which are potent antigen-presenting cells, express c-MIR mRNA or not. Immature DCs were differentiated from monocytes that had been purified from PMBCs by negative selection with anti-CD3, anti-CD7, anti-CD19, anti-CD45RA, anti-CD56, and anti-IgE. 95% of the cells obtained in this way were CD14- CD11c+ (data not shown). The remaining cell fraction from monocyte isolation was collected as PBLs. Immature DCs, monocytes, and PBLs were subjected to RT-PCR analysis. As shown in Fig. 3B, c-MIR mRNA was detected in monocytes and DCs but not in PBLs. The intensity of PCR band of DCs was stronger than that of monocytes, suggesting that DCs express c-MIR mRNA more than monocytes. To confirm that DCs express the authentic c-MIR protein, rabbit polyclonal antibody directed against c-MIR was generated. 1 × 107 immature DCs were labeled with [35S]methionine and [35S]cysteine for 6h, and metabolically labeled cell lysates were immunoprecipitated with anti-c-MIR polyclonal antibody (alpha -c-MIR in Fig. 3C) or rabbit preimmune serum (Cont in Fig. 3C). To verify the full size of the authentic c-MIR protein, the epitope tag sequences (e.g. V5 and His tag) were deleted from the c-MIR expression plasmid, and the resulting expression plasmid was transfected into 293T cells. Transfected 293T cells (293T+c-MIR in Fig. 3C) or nontransfected 293T cells (293T in Fig. 3C) were labeled with [35S]methionine and [35S]cysteine and immunoprecipitated with anti-c-MIR antibody or preimmune serum. As shown in Fig. 3C, this antibody was able to precipitate the proteins of the same molecular weight (marked with asterisks in Fig. 3C) from cell lysates of DCs and transfected 293T cells, but not from nontransfected 293T cell lysates. On the other hand, with preimmune serum, a band corresponding to c-MIR was not detected from any cell lysates. These results indicate that immature DCs express the authentic c-MIR protein. Taken together, these findings suggest that c-MIR might be a functional molecule in DCs.

c-MIR Induces Rapid Endocytosis and Lysosomal Degradation of B7-2-- To reveal the molecular mechanism of B7-2 down-regulation, protein synthesis, degradation, and the trafficking of B7-2 were examined. c-MIR cells were pulse-labeled with [35S]methionine and [35S]cysteine and chased for the indicated periods of time. At the end of the chase periods, pulse-labeled proteins were immunoprecipitated with anti-B7-2 or MHC I antibody and analyzed by SDS-PAGE. Because of the high degree of glycosylation of B7-2, fully matured B7-2 molecules (marked with four small closed triangles in Fig. 4A) obviously migrated slower than the immature form (marked with asterisks in Fig. 4A). In contrast, fully glycosylated MHC I migrated less slowly than the immature form, probably because of the lower degree of glycosylation. In control cells (Cont), the amount of fully matured B7-2 did not decrease significantly up to 6 h. In contrast, in c-MIR cells (c-MIR), at 3 and 6 h, the amount of fully matured B7-2 was significantly reduced compared with control cells (Fig. 4A). To confirm an enhanced degradation of B7-2, B7-2 was treated with PNGase-F to remove glycans, and the treated protein was analyzed by SDS-PAGE. As shown in Fig. 4A, this experiment clearly demonstrated an enhanced degradation of B7-2 by c-MIR. In addition, at 0 h, there was not a significant difference of the intensity of bands between control cells and c-MIR cells, indicating that the down-regulation of B7-2 surface expression is not due to the inhibition of the protein synthesis. Consistent with the observation that c-MIR did not down-regulate MHC I surface expression, degradation of MHC I was not significantly enhanced by c-MIR (Fig. 4A). Rapid degradation of the target molecule was also observed in MIR1 or MIR2-expressing BJAB cells, and this degradation was shown to take place in lysosome (1, 9). Because of the structural and functional similarity of c-MIR to MIR1 and MIR2, the possibility of lysosomal degradation of B7-2 was examined. Treatment of c-MIR cells with bafilomycin A1, which raises endolysosomal pH through the inhibition of the vacuolar H+-ATPase, increased the steady state level of B7-2 protein as judged by immunoblot analysis of whole cell lysate (Fig. 4B). Furthermore, pulse-chase analysis was performed to confirm the inhibition of B7-2 degradation by bafilomycin A1 (Fig. 4C). Control cells and c-MIR cells were pretreated with bafilomycin A1 for 6 h, pulse-labeled with [35S]methionine and [35S]cysteine, chased, and analyzed as in Fig. 4A. Immunoprecipitated B7-2 proteins were treated with PNGase-F and analyzed by SDS-PAGE. As shown in Fig. 4C, pretreatment with bafilomycin A1 inhibited degradation of B7-2. These results demonstrate that c-MIR targets B7-2 for lysosomal degradation.


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Fig. 4.   c-MIR targets B7-2 to lysosomal degradation. A, BJAB cells (Cont) and c-MIR cells (c-MIR) were pulse-labeled with [35S]methionine and [35S]cysteine for 30 min and chased for 1-6 h. At the end of the chase periods, the cells were lysed and immunoprecipitated with anti-B7-2 or anti-MHC I antibody. Each precipitated protein sample was analyzed by SDS-PAGE after treatment with PNGase-F (+) or not (-). B, c-MIR cells were treated with 10 µM bafilomycin A1, an inhibitor of lysosomal degradation, for 2-4 h. At the end of treatment, the cells were subjected to immunoblot analysis with an anti-B7-2 antibody (upper panel) or anti-actin antibody (lower panel). C, c-MIR cells were treated with 10 µM bafilomycin A1 (shown as +) or Me2SO as control (shown as -) for 6 h and subjected to pulse-chase analysis as in Fig. 3A. At the end of the chase periods, the cells were lysed and immunoprecipitated with anti-B7-2, followed by SDS-PAGE analysis after treatment with PNGase-F.

Next, the possibility of enhanced endocytosis of B7-2 was examined; MIR1 and MIR2 do not affect the trafficking of target proteins to the plasma membrane but instead function by altering the endocytic pathway (1, 9, 21). BJAB cells (Cont) and c-MIR cells (c-MIR) were stained with PE-conjugated anti-B7-2 antibody at 4 °C, washed with PBS to remove unbound antibodies, and incubated in complete RPMI medium at 37 °C for 10 and 30 min. After the incubation, uninternalized antibodies were removed with acidic solution, and internalized fluorescence signal was measured by flow cytometry. Even after incubation for 10 min, significant internalized fluorescence was observed in c-MIR cells (shown as the shaded histogram in panel 10 of c-MIR cells in Fig. 5A), but not in the control cells were observed (shown as the shaded histogram in panel 10 of the control cells in Fig. 5A). After incubation for 30 min, there was no obvious increase in internalized fluorescence in c-MIR cells, and significant internalized signals were not yet observed in the control cells (Fig. 5A). These results suggest that c-MIR induces rapid endocytosis of B7-2 that is probably accomplished within 10 min. The same analysis showed that the endocytosis of MHC I was not enhanced in c-MIR cells (data not shown). To confirm c-MIR-induced rapid endocytosis of B7-2, we visualized endocytosed B7-2 molecules by confocal microscopy. The c-MIR cells and control cells were incubated with fluorescein isothiocyanate-labeled anti-B7-2 antibody at 37 °C and examined for the localization of B7-2 molecules. This examination clearly showed internalized B7-2 molecules in the c-MIR cells but not in the control cells (Fig. 5B).


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Fig. 5.   c-MIR induces rapid endocytosis of B7-2. A, BJAB cells (Cont) and c-MIR cells (c-MIR) were stained with PE-conjugated anti-B7-2 antibody at 4 °C, washed with PBS to remove unbound antibodies, and incubated in complete RPMI medium at 37 °C for 10 and 30 min. After the incubation, uninternalized antibodies were removed with acidic solution, and internalized fluorescence was measured by flow cytometry. These results are shown as shaded histograms in panels labeled with 10 and 30, respectively, to their left. To determine the background level of fluorescence, just after being stained with PE-conjugated anti-B7-2 antibody, cell surface-associated antibodies were removed with acidic solution, and a background level was measured by flow cytometry. These results are shown as bold lines in panels labeled 10 or 30. The panels labeled Pre show the level of B7-2 surface expression before stripping of surface-associated antibodies. The results of the staining with isotype control antibody are shown as dotted lines. B, BJAB cells (Cont) and c-MIR cells (c-MIR) were incubated with fluorescein isothiocyanate-labeled anti-B7-2 antibody at 37 °C for 2 h, and then internalized B7-2 molecules were observed with confocal microscopy.

Transmembrane and/or Cytoplasmic Regions of B7-2 Are Involved in Specific Targeting by c-MIR-- Previously, we demonstrated that the transmembrane and cytoplasmic regions of target molecules are sufficient for MIR1 and MIR2-mediated down-regulation by employing CD8 chimeras (2, 21). To see whether the same regions of B7-2 are sufficient for c-MIR-mediated down-regulation, CD8 chimeras were constructed with HLA-A2 and B7-2 molecules. CD8/A2 and CD8/B7-2 chimeras contain the transmembrane and cytoplasmic regions of HLA-A2 and B7-2, respectively, fused to the carboxyl terminus of the extracellular region of CD8alpha (Fig. 6A). These CD8 chimeras were expressed in control BJAB or c-MIR cells, and CD8 surface expression was examined by flow cytometry. Because there is no CD8 expression on the surface of BJAB cells, this approach gives a clear signal for detecting down-regulation of the target molecule. Both CD8 chimeras were expressed efficiently on the surface of control BJAB cells (Fig. 5). On the other hand, whereas CD8/B7-2 chimera was not expressed efficiently on the surface of c-MIR cells, the CD8/A2 chimera was (Fig. 6B). This result confirms the specific targeting by c-MIR and shows that transmembrane and/or cytoplasmic regions of the target molecule are involved in this specificity.


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Fig. 6.   Transmembrane and/or cytoplasmic regions of B7-2 are involved in c-MIR-mediated down-regulation. A, schematic representation of the structure of CD8 chimeras used in this experiment. Extracellular domain (Extra), transmembrane domain (Tm), cytoplasmic domain (Cyto) are indicated. B, BJAB cells (Cont) and c-MIR cells (c-MIR) were transiently transfected with GFP-CD8/A2 or GFP-CD8/B7-2 vector by electroporation. After 24 h, the transfected cells were stained with PE-labeled anti-CD8 antibody, and the surface expression of CD8 chimeras and the expression of GFP were analyzed by two-color flow cytometry. The y axis shows the surface expression of CD8, and the x axis shows the expression of GFP.

c-MIR Functions as an E3 Ubiquitin Ligase-- c-MIR contains a BKS-PHD domain similar to MIR1 and MIR2. The BKS-PHD domain of MIR1 and MIR2 was shown to be a functional domain for E3 ubiquitin ligase (6, 7). To verify the E3 ubiquitin ligase activity of the BKS-PHD domain of c-MIR, the BKS-PHD domain of MIR1 was replaced with that of c-MIR, and the resulting chimeric protein, termed MIR1/c-MIR, was subjected to flow cytometry (Fig. 7A). Another chimera containing a mutated BKS-PHD domain of c-MIR, termed MIR1/c-MIR(Cys right-arrow Ser), was also included (Fig. 7A). In this mutant, cysteines at positions 80, 83, 123, and 125, which are putative zinc-binding residues, were mutated to serine. These chimeras and MIR1 were expressed in A7 cells, and MHC I surface expression was examined by two-color flow cytometry. As expected, MIR1/c-MIR down-regulated MHC I surface expression efficiently (Fig. 7B). This down-regulation was also dependent on BKS-PHD domain; MIR1/c-MIR(Cys right-arrow Ser) was not able to down-regulate the surface expression of MHC I. These results strongly suggest that c-MIR has E3 ubiquitin ligase activity. To confirm this, an in vitro auto-ubiquitination assay was performed. The wild or mutant type of the BKS-PHD domain of c-MIR was fused to the carboxyl terminus of GST, and purified GST fusion proteins were subjected to auto-ubiquitination assay in a mixture of ATP, free ubiquitin, E1, and E2 (UbcH5a). A clear polyubiquitinated form of the wild type BKS-PHD domain-containing GST fusion protein (GST-c-MIR) was observed, but GST alone (GST) or mutated BKS-PHD domain-containing GST fusion protein (GST-c-MIR(Cys right-arrow Ser)) did not have this form (Fig. 7C). GST-c-MIR(Cys right-arrow Ser) has the same mutation as MIR1/c-MIR(Cys right-arrow Ser) in the BKS-PHD domain. This result confirmed the E3 ubiquitin ligase activity of the BKS-PHD domain of c-MIR. To test whether B7-2 can be ubiquitinated by c-MIR, B7-2 was co-expressed with HA-tagged ubiquitin and either wild type c-MIR (c-MIR) or mutant type c-MIR whose BKS-PHD domain was mutated (termed c-MIR(Cys right-arrow Ser)) as done in the case of MIR1/c-MIR(Cys right-arrow Ser). These cell lysates were precipitated with anti-B7-2 antibody and subjected to immunoblot analysis with anti-HA or B7-2 antibody. With co-expression of c-MIR, a clear polyubiquitinated form of B7-2 was detected by HA and B7-2 blots but not without expression of c-MIR (Cont) or with co-expression of c-MIR(Cys right-arrow Ser) (Fig. 7D). Taken together, these findings confirm that c-MIR functions as a E3 ubiquitin ligase for the B7-2 co-stimulatory molecule through its BKS-PHD domain.


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Fig. 7.   c-MIR is an E3 ubiquitin ligase for B7-2. A, schematic representation of the structure of MIR-1 and its chimeras used in this experiment. The BKS-PHD domain of MIR1 (8-57 amino acids) was replaced by that of c-MIR (78-137 amino acids) to construct MIR1/c-MIR. In addition, cysteines 80, 83, 123, and 126 in the BKS-PHD domain of c-MIR were mutated to serine and also used to replace the c-MIR BKS-PHD domain as above. This chimera was named MIR1/c-NIR(Cys right-arrow Ser). Transmembrane domain (Tm) and BKS-PHD domain are indicated. CC CC HC CC is the consensus amino acid sequence of the BKS-PHD domain. B, GFP-MIR1, GFP-MIR1/c-MIR and GFP-MIR1/c-MIR(Cys right-arrow Ser) vectors were transfected in A7 cells, and surface expression of MHC I was analyzed as in Fig. 2A. C, the BKS-PHD domain of c-MIR (78-137 amino acids) was fused to GST and subjected to in vitro auto-ubiquitination assay. Mutant BKS-PHD domain that has the same mutations as MIR1/c-MIR(Cys right-arrow Ser) was also included. GST alone (GST), the GST-BKS-PHD domain of c-MIR (GST-c-MIR), and the GST-mutant BKS-PHD domain of c-MIR (GST-c-MIR(cys>ser)) were incubated in ubiquitination buffer (details under "Experimental Procedures") for 3 h. The incubated samples were probed with anti-ubiquitin antibody (alpha -Ubi). D, 293T cells were co-transfected with HA-tagged ubiquitin expression plasmid, pEF-B7-2, and one of the following plasmid DNAs: pEF (Cont), pEF-c-MIR (c-MIR), and pEF-c-MIR(Cys right-arrow Ser) (c-MIR(cys>ser)). c-MIR(Cys right-arrow Ser) has the same mutations as MIR1/c-MIR(Cys right-arrow Ser) shown in A. Each co-expressed protein was immunoprecipitated with anti-B7-2 (top and middle panels) and probed with anti-HA antibody (top panel) or anti-B7-2 antibody (middle panel). Whole cell lysates were probed with anti-His antibody to show the comparable expression of c-MIR or c-MIR(Cys right-arrow Ser) in each cell lysate (bottom panel). IP, immunoprecipitation; IB, immunoblot.

Ubiquitination Is Necessary for c-MIR-mediated Down-regulation of B7-2 Surface Expression-- We earlier showed c-MIR as a modulator of B7-2 surface expression and a novel E3 ubiquitin ligase for B7-2. However, it was still unknown whether or not ubiquitination was really necessary for down-regulation of B7-2 surface expression. To clarify this point, c-MIR(Cys right-arrow Ser), a ubiquitination dead mutant, was transiently expressed in BJAB cells by electroporation and examined by two-color flow cytometry. As shown in Fig. 8B, c-MIR(Cys right-arrow Ser) did not down-regulate the surface expression of B7-2 at all. We subsequently constructed a mutant B7-2 molecule that was no longer ubiquitinated by c-MIR. It has been shown that MIR2 mediates the ubiquitination of lysine residues located at the cytoplasmic tail of targets (6). This report led us to test whether this is the case with c-MIR. For this examination, the CD8 chimera used in Fig. 6 was employed. All of the lysine residues located at the B7-2 cytoplasmic region of the CD8/B7-2 chimera were mutated to arginine by PCR-based mutagenesis and modified to encode a FLAG epitope tag at its amino terminus. This chimera was termed CD8-B7(Lys right-arrow Arg). Furthermore, the CD8/B7-2 chimera was modified to encode a FLAG epitope tag at its amino terminus, and the resulting modified chimeric protein was termed CD8-B7. Each FLAG-tagged CD8 chimera was co-expressed with HA-tagged ubiquitin and c-MIR in 293T cells and subjected to the same experiment as performed in Fig. 7D. HA and FLAG blots clearly showed that CD8-B7(Lys right-arrow Arg) was no longer ubiquitinated by c-MIR (Fig. 8C). To see whether ubiquitination is linked to down-regulation of target surface expression, CD8-B7(Lys right-arrow Arg) and CD8-B7 were expressed in BJAB cells (control cells) or c-MIR cells, and the surface expression level of CD8 was compared. Flow cytometry analysis showed that the expression of CD8-B7(Lys right-arrow Arg) was not inhibited in c-MIR cells (Fig. 8D), suggesting that the ubiquitination of cytoplasmic lysine residues of B7-2 is necessary for down-regulation.


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Fig. 8.   Ubiquitination is necessary for the down-regulation of B7-2 surface expression. A, schematic representation of the structure of c-MIR and its mutant used in this experiment. B, BJAB cells were transfected with GFP-c-MIR or GFP-c-MIR(Cys right-arrow Ser) vector, and the surface expression of B7-2 and the expression of GFP were analyzed by two-color flow cytometry. C, all of the lysine residues in the cytoplasmic tail of CD8/B7-2 were mutated to arginine by PCR-based mutagenesis to construct CD8/B7(Lys right-arrow Arg). CD8/B7-2 and CD8/B7(Lys right-arrow Arg) were modified to encode a FLAG epitope tag at their amino termini and termed CD8-B7 and CD8-B7(Lys right-arrow Arg), respectively. CD8-B7-2 and CD8-B7(Lys right-arrow Arg) were co-expressed in 293T cells with HA-tagged ubiquitin and c-MIR, and their ubiquitination status was analyzed as in Fig. 7D. Each expressed protein was immunoprecipitated with anti-FLAG antibody and probed with anti-HA (left upper panel) or with anti-FLAG antibody (right upper and lower panels). The right upper and lower panels show the results of the same membrane, which was probed with anti-FLAG antibody. The upper part was exposed for a longer time than the lower part because of the weak signal of ubiquitinated FLAG-CD8 chimera (shown as ub-FLAG-CD8 chimera). To show the same expression of c-MIR, each whole cell lysate was probed with anti-His antibody (left lower panel). D, BJAB cells (Cont) and c-MIR cells (c-MIR) were transfected with GFP-CD8-B7 or GFP-CD8-B7(Lys right-arrow Arg) vector, and the surface expression of CD8 and the expression of GFP were analyzed by two-color flow cytometry. IP, immunoprecipitation; IB, immunoblot.

Specific Ubiquitination and Down-regulation of B7-2 through Molecular Interaction-- The possible molecular interaction of c-MIR and B7-2 was then examined, because E3 ubiquitin ligase has been shown to function through binding to targets (22, 23). c-MIR and CD8-B7 were expressed together or individually in 293T cells, and these cell lysates were analyzed by immunoprecipitation and immunoblot analysis as indicated. Only when c-MIR and CD8-B7 were expressed together, a specific band corresponding to c-MIR was detected, demonstrating the molecular interaction of both proteins (Fig. 9A). It has been proposed that the transmembrane regions of MIR1 and MIR2 confer specific targeting through binding to the target protein transmembrane region (6, 24). If so, c-MIR(Cys right-arrow Ser), which has intact transmembrane domains, is expected to be able to associate with the target. To test this hypothesis, c-MIR(Cys right-arrow Ser) was subjected to the same assay as performed in Fig. 9A. As shown in Fig. 9B, a clear band corresponding to c-MIR(Cys right-arrow Ser) was detected only when both c-MIR(Cys right-arrow Ser) and CD8-B7 were expressed. These results indicate that both c-MIR and c-MIR(Cys right-arrow Ser) were able to bind to CD8-B7.


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Fig. 9.   Molecular interaction of c-MIR and CD8-B7. A, c-MIR and CD8-B7 were expressed in 293T cells either together or individually as indicated. The amount of each transfected DNA was adjusted by adding empty vector DNA. 48 h after transfection, the expressed proteins were immunoprecipitated with anti-FLAG antibody and probed with anti-V5 antibody that recognizes c-MIR (top panel). To show equal expression of CD8 chimeras, the same precipitated samples were probed with anti-FLAG antibody (middle panel). Whole cell lysate was probed with anti-V5 antibody to show equal expression of c-MIR. B, the same analysis was performed by using c-MIR(Cys right-arrow Ser). C, CD8-B7 was co-expressed with c-MIR or c-MIR(Cys right-arrow Ser) in A7 cells. CD8-B7 was visualized by staining with anti-CD8 monoclonal antibody and Alexa 568-conjugated secondary antibody. c-MIR and c-MIR(Cys right-arrow Ser) were visualized by staining with anti-His polyclonal antibody and Alexa 488-conjugated secondary antibody. IP, immunoprecipitation; IB, immunoblot.

Next, to confirm the molecular interaction, we examined where they associate with each other by immunofluorescence microscopy. For this purpose, we used A7 cells in which the relatively large cytoplasmic content facilitates the examination of subcellular localization. c-MIR or c-MIR(Cys right-arrow Ser) was co-expressed in A7 cells with CD8-B7 and stained with anti-His antibody for c-MIR and c-MIR(Cys right-arrow Ser) and with anti-CD8 antibody for CD8-B7. As shown in Fig. 9C, both c-MIR and c-MIR(Cys right-arrow Ser) were co-localized with CD-B7. c-MIR was localized mainly in the perinuclear region. On the other hand, c-MIR(Cys right-arrow Ser) was localized in both the plasma membrane and perinuclear regions. These results confirmed the molecular interaction of B7-2 with c-MIR or c-MIR(Cys right-arrow Ser). During these experiments, we found that a complex of c-MIR(Cys right-arrow Ser) and CD8-B7 was more easily detected than that of wt-c-MIR and CD8-B7, probably because of the instability of wt-c-MIR/CD8-B7 complex. Consistent with this explanation, the amount of the high molecular form of CD8-B7, a probably matured form, was drastically reduced by co-expression of c-MIR compared with the single expression of CD8-B7 (Fig. 9A), and the intensity of plasma membrane staining of CD8-B7 was drastically reduced by co-expression of c-MIR (Fig. 9C).

To see whether specific down-regulation of B7-2 is due to molecular interaction, CD8/A2 used in Fig. 6 was employed for further examination because the surface expression of CD8/A2 chimera was not down-regulated in c-MIR cells (Fig. 6B). CD8/A2 was modified to encode a FLAG epitope tag at its amino terminus, termed CD8-A2, and CD8-A2 or CD8-B7 was co-expressed with c-MIR(Cys right-arrow Ser) in 293T cells, and the possible interaction was examined by immunoprecipitation and immunoblotting analysis. In this study, we employed c-MIR(Cys right-arrow Ser) because of easy detection of c-MIR-CD-8-B7 complexes. As shown in Fig. 10A, c-MIR(Cys right-arrow Ser) was efficiently co-precipitated with CD8-B7, but not with CD8-A2, suggesting that molecular interaction is necessary for specific down-regulation by c-MIR. To clarify whether the molecular interaction is linked to specific ubiquitination of targets, CD8-A2 and CD8-B7 were subjected to the same analysis as performed in Fig. 8C. As shown in Fig. 10B, CD8-B7, but not CD8-A2, was efficiently ubiquitinated. These results demonstrate that a molecular interaction is necessary for c-MIR-mediated ubiquitination and the following down-regulation of the target molecule.


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Fig. 10.   Specific ubiquitination through binding to the target. A, CD8/A2 used in Fig. 5 was tagged with FLAG epitope at its amino terminus. FLAG-CD8-A2 (CD8-A2) and FLAG-CD8-B7-2 (CD8-B7) were co-expressed with c-MIR(Cys right-arrow Ser) in 293T cells. Cell lysate was immunoprecipitated with anti-HA or FLAG antibody as indicated and probed with anti-V5 (top panel) or anti-FLAG antibody (middle panel). Each whole cell lysate was probed with anti-V5 antibody (bottom panel). B, CD8-A2 or CD8-B7 was co-expressed in A7 cells with HA-tagged ubiquitin and c-MIR, and the ubiquitination status of each molecule was analyzed as in Fig. 8C. IP, immunoprecipitation; IB, immunoblot.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have identified a functional homolog of KSHV MIR1 and MIR2 in the human genome. This functional human homolog has been designated as c-MIR. c-MIR targets the B7-2 co-stimulatory molecule to lysosomal degradation through the enhancement of endocytosis, eventually leading to down-regulation of B7-2 surface expression. Moreover, the binding of c-MIR to B7-2 and the following ubiquitination of the B7-2 cytoplasmic tail are necessary for down-regulation of B7-2 surface expression. MIR1, MIR2, and c-MIR share the same secondary structure, a similar BKS-PHD domain, and the same positioning of the BKS-PHD domain, suggesting that these molecules belong to the same class of E3 ubiquitin ligase. Although the physiological role of c-MIR is as yet unknown, our findings suggest some attractive hypotheses that are discussed below.

The first hypothesis is that c-MIR might be a novel regulator for antigen presentation because of its unique targeting property. The B7-2 co-stimulatory molecule supports MHC class II-mediated antigen presentation to effector T cells on the surface of antigen presenting cells (e.g. dendritic cells and B cells) and has been shown to be one of the modulators for immune synapse formation (25, 26) between T cells and antigen presenting cells. The formation of an immunological synapse has been demonstrated to potentiate T cell activation (27, 28). Furthermore, co-stimulatory signaling has been implicated in the progression of autoimmune diseases (29-31). Hence, c-MIR-mediated modulation of B7-2 surface expression is expected to regulate the status of T cell activity and immunity. To establish an animal model and test this attractive hypothesis, we cloned mouse c-MIR from murine B cell lines. Mouse c-MIR showed exactly the same function,2 suggesting that mice also have a similar system. This mRNA was detected by RT-PCR in most tissues except in the thymus.2 This preliminary finding may still be consistent with our hypothesis, because B7-2 is expressed abundantly in the thymus. If c-MIR is expressed abundantly in the thymus, B7-2 would be expressed inefficiently. A detailed examination of the profile of tissue distribution and the expression status of c-MIR in either a physiological or pathological setting and genetically modified mice are necessary to support our hypothesis.

Another hypothesis is related to the origin of MIR1 and MIR2. The KSHV genome encodes many homologs of human genes such as viral interleukin-6, viral macrophage inflammatory protein, and vBcl-2 (15). In Fig. 7B, we showed that the BKS-PHD domain of MIR1 could be replaced with that of c-MIR without alteration of the function of MIR1. These findings suggest that at least the functional domains of MIR1 and MIR2 might have originated in the host genome. Moreover, the homologs of MIR1 and MIR2 have been identified in simian and bovine gamma -herpesviruses and in swinepox virus (8), suggesting that these proteins might be derived from a common host predating divergence of these species. This hypothesis might be extended to other viral immune regulatory proteins (e.g. US2,11 of human cytomegalovirus). Herpesviruses have many immune regulatory proteins that down-regulate MHC I surface expression (32, 33). So far, there are no reports describing functional host homologs of these proteins. In the case of MIR1 and MIR2, the existence of a unique functional domain, the BKS-PHD domain, made it easy to identify functional homolog candidates. For other viral immune regulatory proteins, however, this might prove difficult because there are no well known domains in these proteins so far.

At present, several classes of E3 ubiquitin ligase have been discovered (23). Catalytic domains of E3 ubiquitin ligase are classified into two major groups: HECT (homologous to E6-AP carboxyl terminus) or RING domains. HECT and RING domains are critical for their E3 ubiquitin ligase activities, because they recruit E2 and transfer ubiquitin to target molecules. In this study, we showed that a mammalian BKS-PHD domain, which is a subclass of the PHD domain whose viral version is represented by the KSHV MIR proteins, was essential for ubiquitination in vivo and able to function in cooperation with UbcH5a/E2 in vitro (Fig. 7, C and D). Further, this mammalian BKS-PHD domain was able to function in the context of KSHV MIR1 (Fig. 7B). Because the sequence of the BKS-PHD domain resembles the RING domain consensus sequence CX2CX9-39CX1-3HX2-3(C/H)X2CX4-48CX2C, we would like to propose that the BKS-PHD domain is a novel subclass of RING domain in mammals as well as in viruses. In this regard, the E3 ubiquitin ligase activity of the mammalian PHD domain has been reported in mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (14). There are several mammalian BKS-PHD domain-containing proteins in the public protein data base whose functions are still unknown. It will be important to analyze these hypothetical proteins and their targets for E3 ubiquitin ligase activity.

Our findings show that the mechanism of c-MIR-mediated down-regulation is strikingly similar to that of KSHV MIR1 and MIR2. c-MIR, like MIR1 and MIR2, ubiquitinates a target molecule and leads to rapid endocytosis and translocation to lysosome, followed by rapid degradation of a target molecule. So far, they only differ with regard to their specific target molecules. It will be important to understand how they specifically target different molecules. In this connection, it has been reported that an MIR1 chimera containing the transmembrane domain of MIR2 is able to down-regulate the MIR2 targets B7-2 and ICAM-1 (24), suggesting that different targeting is achieved by the binding property of the transmembrane domains of MIR1 and MIR2. Although both MIR2 and c-MIR were able to target B7-2 (21, 34), there was no significant homology between their transmembrane domains. Further detailed analysis is necessary to reveal how these E3 ubiquitin ligases regulate targeting specificity.

    ACKNOWLEDGEMENTS

We thank J. U. Jung for sharing materials, R. E. Means for technical advice, D. Bohmann for the HA-Ubi expression plasmid, G. B. Cohen for CD8alpha cDNA and HLA-A2 cDNA, and W. E. Johnson and R. H. Florese for critical reading of this manuscript.

    FOOTNOTES

* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to S. I.) and also by grants from the Uehara Memorial Foundation and the Hyogo Science and Technology Association (to S. I.).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.

Dagger These authors contributed equally to this work.

§ To whom all correspondence should be addressed. Tel.: 81-78-382-5501; Fax: 81-78-382-5519; E-mail: ishido@med.kobe-u.ac.jp.

Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M211285200

2 E. Goto, S. Ishido, Y. Sato, S. Ohgimoto, K. Ohgimoto, M. Nagano-Fujii, and H. Hotta, unpublished data.

    ABBREVIATIONS

The abbreviations used are: KSHV, Kaposi's sarcoma-associated herpesvirus; PHD, plant homeodomain; MHC, major histocompatibility complex; HLA, human leukocyte antigen; PNGase, peptide N-glycanase; HA, hemagglutinin; GST, glutathione S-transferase; ICAM-1, intercellular adhesion molecule 1; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; RT, reverse transcriptase; GAPDH, glyceraldehye-3-phosphate dehydrogenase; GFP, green fluorescent protein; DC, dendritic cell; PBMC, peripheral blood mononuclear cell; PBL, peripheral blood lymphocyte; PBS, phosphate-buffered saline; PE, phycoerythrin.

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ABSTRACT
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RESULTS
DISCUSSION
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