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. 2011 Oct 28;286(43):37168-80.
doi: 10.1074/jbc.M110.204040. Epub 2011 Sep 6.

Ubiquitin-mediated regulation of CD86 protein expression by the ubiquitin ligase membrane-associated RING-CH-1 (MARCH1)

Kathleen Corcoran  1 , Maurice Jabbour, Candida Bhagwandin, Martin J Deymier, Debra L Theisen, Lonnie Lybarger
Affiliations

Ubiquitin-mediated regulation of CD86 protein expression by the ubiquitin ligase membrane-associated RING-CH-1 (MARCH1)

Kathleen Corcoran et al. J Biol Chem. .

Abstract

The activation of naïve T cells requires antigen presentation by dendritic cells (DCs), and the process of antigen presentation is regulated over the course of DC maturation. One key aspect of this regulation is the cell surface up-regulation upon DC maturation of peptide·MHC-II complexes and the costimulatory molecule CD86. It is now clear that these critical induction events involve changes in ubiquitin-dependent trafficking of MHC-II and CD86 by the E3 ligase membrane-associated RING-CH-1 (MARCH1). Although ubiquitin-dependent trafficking of MHC-II has been well characterized, much less is known regarding the post-transcriptional regulation of CD86 expression. Here, we examined the physical and functional interaction between CD86 and MARCH1. We observed that CD86 is rapidly endocytosed in the presence of MARCH1 followed by lysosome-dependent degradation. Furthermore, we found that the association between CD86 and MARCH1 was conferred primarily by the transmembrane domains of the respective proteins. In contrast to MHC-II, which has a single, conserved ubiquitin acceptor site in the cytosolic domain, we found that multiple lysine residues in the cytosolic tail of CD86 could support ubiquitination consistent with the relative lack of sequence conservation across species within the CD86 cytosolic domain. These findings suggest that MARCH1 recruits multiple substrates via transmembrane domain-mediated interactions to permit substrate ubiquitination in the face of diverse cytosolic domain sequences.

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Figures

FIGURE 1.
FIGURE 1.
Rapid loss of cell surface CD86 in presence of MARCH1. A, flow cytometry was used to examine the surface levels of CD80 and CD86 on DC2.4 cells +/− MARCH1 (top row) and CD80, CD86, and MHC-II on MJDC cells +/− MARCH1 (middle and bottom rows). In some cases, MJDC cells were treated with 100 units/ml IFN-γ for 18 h prior to staining to increase MHC-II synthesis. Gray histograms represent staining using an isotype control antibody. Control shown for DC2.4 cells represents staining of the parental cell line, whereas Control for MJDC cells represents staining of cells transduced with the empty retroviral vector. All histograms for MJDC were gated on GFP+ (either vector-only or MARCH1-expressing) cells. B and C, DC2.4 cells +/− MARCH1 were stained with unlabeled CD86 antibody (GL1) at 4 °C for 30 min. After washing, cells were incubated in culture medium at 37 °C for the indicated time points and then transferred to ice. Cells were then incubated with a fluorochrome-conjugated secondary antibody, washed with D-PBS, fixed with 1% paraformaldehyde, and analyzed by flow cytometry. For each time point, the mean fluorescence intensity (MFI) value for CD86 surface expression (of triplicate samples) was normalized relative to the mean fluorescence intensity value for time 0 and plotted as percent (%) surface CD86 remaining ±S.E. (**, p < 0.01). Internalization data are representative of at least three independent experiments each for B and C; error bars are not apparent on the graphs because their ranges are smaller than the symbols used. PE, phycoerythrin.
FIGURE 2.
FIGURE 2.
MARCH1 associates with CD86 and promotes ubiquitination and turnover. A, whole cell lysates from DC2.4 cells +/− MARCH1 WT or MARCH1 W104A were immunoblotted for MARCH1, CD86, CD80, and actin (loading control). Baf A treatment (0.1 μm) for 3 h was also performed where indicated. B, CD86 was immunoprecipitated from digitonin lysates of DC2.4 cells (+/− MARCH1 WT or W104A), and samples were resolved by SDS-PAGE and blotted for MARCH1 and CD86. C, CD86 was immunoprecipitated (IP) from cell lysates of DC2.4 cells +/− MARCH1 WT or W104A. Precipitates were untreated (upper panel) or N-glycosidase F (endoF)-treated (middle panel) and then immunoblotted for ubiquitin (Ub) and CD86 (lower panel; non-treated samples). D, DC2.4 cells +/− MARCH1-HA were either left untreated or treated with 0.1 μm Baf A for 3 h. cells were then stained using indirect double label immunofluorescence with rabbit anti-HA tag (6E2) and rat anti-CD86 (GL-1). Antigens were visualized by confocal microscopy, and images shown represent a single optical section (left panels). Detector settings were identical for all images, and images shown are representative of three independent experiments. The right panel shows flow cytometric analysis of surface CD86 expression of cells treated as above. E, DC2.4 cells +/− MARCH1 WT or W104A were pulse-labeled with [35S]Met/Cys and chased for the indicated time points with unlabeled Cys/Met. CD86 immunoprecipitation was performed from cell lysates followed by N-glycosidase F treatment, SDS-PAGE, and autoradiography. The signal intensity for each band at each chase time point was determined and normalized to the signal intensity value of the band corresponding to time 0 and plotted as percentage (%) of time 0. The graph represents data from three replicates (±S.E.; **, p < 0.01). F, pulse-chase was performed as in D with the exception that cells were also treated with Baf A (0.1 μm) during the chase period. The results shown are representative of three independent experiments.
FIGURE 3.
FIGURE 3.
Domains of MARCH1 and CD86 that mediate their interaction. A, co-immunoprecipitation (IP) of CD86 was performed using lysates from DC2.4 parent cells or cells stably expressing MARCH1 WT, ΔN1–121, ΔC229–279, or ΔC257–279. Whole cell lysates (middle panels) and CD86 precipitates (right panels) were blotted for CD86 and MARCH1. Note that for MARCH1 ΔN1–121, one-third of the CD86 precipitate was loaded relative to MARCH1 WT to achieve comparable signal intensity; this mutant is significantly more stable than WT. B, flow cytometry was used to examine the surface expression of the indicated chimeras in the presence of MARCH1. Panel i, a fibroblast cell line derived from β2m−/− Kb−/− Db−/− mice (3KO cells) +/− MARCH1 (GFP+) were transduced with the chimeras consisting of the human β2m (ectodomain) fused to the transmembrane and cytosolic regions of murine CD86 (Tβ2m) or with a β2m chimera lacking the cytosolic tail of CD86 (Tβ2m.ΔCT). Panel ii, 3KO cells expressing MARCH1 were transduced with either Tβ2m or Tβ2m lysineless (K-less) (lysine-to-arginine mutation of all five lysines in the tail of mouse CD86). Panel iii, DC2.4 cells were transduced with MARCH1 and either with H2-Ld or a chimera comprising the H2-Ld ectodomain fused to the transmembrane and cytosolic regions of human CD86. PE, phycoerythrin.
FIGURE 4.
FIGURE 4.
Transmembrane and cytosolic regions of CD86 confer association with MARCH1, leading to ubiquitination. A, top panel, cell lysates from 3KO cells expressing MARCH1 and Tβ2m were blotted to reveal the steady-state levels of Tβ2m. Bottom panel, Tβ2m precipitates were resolved by SDS-PAGE and blotted for ubiquitin (Ub) and Tβ2m. B, blots of lysates from 3KO cells expressing Tβ2m, Tβ2m.ΔCT, and Tβ2m.Tpn were probed with the indicated antibodies (left panel). Right panel, the indicated chimeras were precipitated from 3KO cells co-expressing MARCH1 and blotted as indicated. C, bone marrow-derived DCs generated from CD86−/− mice were infected using lentivirus vectors expressing either GFP (vector only), GFP and CD86 WT, or GFP and CD86.ΔCT (lacking the cytosolic region of CD86). 3 days postinfection, cells were harvested and either left untreated (No LPS) or treated with 100 ng/ml LPS for 18 h. Flow cytometry was used to analyze the surface expression of CD86. The histogram on the left represents GFP expression upon which the CD86 histograms are gated. Data are representative of two independent experiments. IP, immunoprecipitation.
FIGURE 5.
FIGURE 5.
Mapping domains of CD86 required for sensitivity to MARCH1. A, map indicating the domains of various CD86/CD80 chimeric molecules. CD86.ΔCT corresponds to residues 1–245; CD86/80 TM corresponds to residues 1–222 of CD86, 212–233 of CD80, and 241–286 of CD86; CD86/80 tail corresponds to residues 1–241 of CD86 and 233–270 of CD80; CD86/80 TM+tail corresponds to residues 1–222 of CD86 and 212–270 of CD80. B, fibroblast cells (WT3) were co-transfected with bicistronic vectors co-expressing MARCH1 (WT or W104A) and GFP as a reporter and either CD86 or CD80. Gray histograms represent CD86 staining (background) of the parental WT3 cell line. Histograms are gated on GFP+ (MARCH1-expressing) cells. C, flow cytometry was used to analyze the surface expression of CD86 on the panel of CD86/CD80 chimeras after stable expression of each construct following transduction of WT3 cells with retroviral vectors. Gray-colored histograms represent isotype control staining. D, the indicated chimeric molecules were precipitated from WT3 cells co-expressing MARCH1 and blotted as indicated. IP, immunoprecipitation. PE, phycoerythrin.
FIGURE 6.
FIGURE 6.
Multiple lysine residues in cytosolic domain of CD86 can support MARCH1-mediated ubiquitination. A, single letter representation of the TM (highlighted in gray) and the cytosolic domain sequences of murine CD80 and CD86. Numbers below each sequence represent the predicted start of the cytosolic region. Lysine residues are indicated (and numbered for CD86), and bars under the CD80 sequence show the location for two blocks of residues that were mutated to lysines (HRS in CD80 3K and RE in CD86/CD80 tail RE-KK). B, comparison of CD86 cytosolic domain lysine mutants for sensitivity to MARCH1. The numbering indicated above each histogram represents the position of lysine residues mutated in the CD86 cytosolic tail relative to the predicted end of the CD86 transmembrane domain. WT3 cells were co-transfected with the indicated CD86 constructs and a bicistronic GFP reporter vector (+/− MARCH1). Flow cytometry was used to examine the surface expression of each CD86 construct +/− MARCH1. All histograms are gated on GFP-expressing cells. The most relevant mutants are shown; plots from all mutants tested are shown in supplemental Fig. S2. The right panel represents a graph of the data from the plots shown here and in supplemental Fig. S2. The geometric mean fluorescence intensity of CD86 staining for each sample was used to determine the percentage (%) of control. This was calculated for each CD86 mutant by comparing the mean fluorescence intensity (MFI) of that mutant +/− MARCH1 as follows: ((+MARCH1 MFI)/(no MARCH1 MFI)) × 100. Data are representative of three independent experiments. C, similar to B with CD86/80 tail construct (see diagram in Fig. 5) with RE mutated to KK (see sequence map in A). D, similar to B with CD80 WT or CD80 construct with three lysine residues added to its cytosolic tail at positions 6, 7, and 8 (underlined in A). In C and D, constructs were co-transfected into WT3 cells +/− MARCH1. Histograms are gated on GFP+ (MARCH1-expressing) cells. mt, mutant.
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