Immunology and Cell Biology (2014) 92, 527–534 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14 www.nature.com/icb
ORIGINAL ARTICLE
LAPTM5 promotes lysosomal degradation of intracellular CD3f but not of cell surface CD3f Yohei Kawai1, Rika Ouchida1, Sho Yamasaki2, Leonard Dragone3, Takeshi Tsubata5 and Ji-Yang Wang4,5 The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3f degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3f but not of the cell surface CD3f associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3f suggests that LAPTM5 targets CD3f in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3f can be transported to and degraded in the lysosome by LAPTM5. A CD3f YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3f, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3f is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3f in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3f can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3f dependent on TCR signaling, and LAPTM5, which targets intracellular CD3f independent of TCR signaling. Immunology and Cell Biology (2014) 92, 527–534; doi:10.1038/icb.2014.18; published online 18 March 2014
T lymphocytes, which have a central role in immune regulation, recognize processed antigens in the context of major histocompatibility complex by virtue of their T cell receptor (TCR). The TCR is a multi-subunit complex composed of the variable antigenbinding TCRab or gd chains that are associated with the invariant signal-transducing CD3ge, de- and zz-subunits.1 TCR signaling must be strictly controlled to allow for normal T-cell development and optimal activation. One important mechanism for regulating the TCR signal is the modulation of its cell surface expression.2 Cell surface TCR levels are the result of a dynamic equilibrium among at least the following four processes: the assembly of the newly synthesized TCR and its transport to the cell surface; internalization of the surface TCR; recycling of the internalized TCR back to the cell surface; and degradation of the internalized TCR. Modulation of any of these processes would affect the steady-state TCR levels.3,4 The TCR complex is initially assembled in the endoplasmic reticulum (ER), where CD3e dimerizes with either CD3g or CD3d and then these dimers associate with TCRa and b-chains.5–7 It has been suggested that only when all chains other than CD3z are assembled is the resulting ade–bge complex exported to the Golgi apparatus, where the CD3z homodimer is bound to form the fully assembled TCR complex.8–10 This stepwise assembly and transport are
known to be regulated by ER retention motifs11–14 and/or endocytic recognition sequences containing di-leucine or tyrosine.15–17 These functional ER retention and endocytic recognition motifs are present in all CD3 subunits other than CD3z, and all of them must be masked by association with other subunits before transport to the plasma membrane.11 The unassembled TCR subunits in the ER are rapidly degraded in the proteasome by an ER-to-cytosol transport mechanism.18 Partially assembled TCR in the Golgi, on the other hand, is degraded in lysosomes unless association of the CD3z homodimer masks di-leucine- and/or tyrosine-based motifs in CD3g, d- and e-chains.16,19,20 After transport to the cell surface, levels of the fully assembled TCR are also dynamically controlled. In CD4 þ CD8 þ double-positive (DP) thymocytes, major histocompatibility complex class II-CD4 interaction and the resultant activation of Lck, a Src family tyrosine kinase, have an important role in maintaining a 10-fold lower expression of TCR compared with peripheral T cells.21–23 Lckmediated phosphorylation of CD3 chains results in TCR endocytosis, recruitment of the Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl, and ubiquitination of CD3z and its subsequent lysosomal degradation.24,25 Accordingly, both SLAP- and c-Cbl-knockout (KO) mice exhibited higher TCR levels on DP
1Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan; 2Division of Molecular Immunology, Research Center for Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 3National Jewish Health Colorado Children’s Hospital, University of Colorado Denver, School of Medicine, Denver, CO, USA; 4Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, China and 5Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Correspondence: J-Y Wang, Department of Immunology, Shanghai Medical College, Fudan University, 130 Dong’an Road, Shanghai 200032, China. E-mail: [email protected] Received 24 November 2013; revised 4 February 2014; accepted 17 February 2014; published online 18 March 2014
LAPTM5 targets intracellular CD3z Y Kawai et al 528
thymocytes due to decreased degradation of the internalized TCR in the lysosome and its increased recycling back to the cell surface.24,25 Previously we have reported that the lysosomal protein LAPTM5 negatively regulates TCR expression by interacting with CD3z and promoting its lysosomal degradation.26 Compared with wild-type (WT) mice, LAPTM5 KO mice show elevated TCR levels on DP thymocytes and antigen-stimulated peripheral T cells. Consequently, T-cell activation is enhanced, with elevated recall responses and prolonged delayed-type hypersensitivity reactions. Furthermore LAPTM5-deficient B cells express higher levels of the BCR compared with WT B cells after antigen stimulation, and the mutant mice show elevated antibody production to foreign antigens and also produce autoantibodies as they age. Therefore, LAPTM5 is a common negative regulator of TCR and BCR expression and T- and B-cell activation.27 Despite the immunological significance of LAPTM5, the molecular mechanism by which it mediates TCR and BCR degradation remains largely unknown. In the present study, we have explored the mechanisms of LAPTM5-mediated CD3z degradation in T cells. By using T cells lacking CD3z and reconstituted with exogenous CD3z, we show that LAPTM5 targets CD3z in the Golgi and promotes its lysosomal translocation, while having little effect on the cell surface CD3z associated with the TCR complex. We also provide genetic evidence that LAPTM5 promotes CD3z degradation through a pathway distinct from SLAP/c-Cbl. These results reveal a previously unidentified mechanism that controls TCR levels. RESULTS LAPTM5 promotes the lysosomal translocation of intracellular CD3f but does not affect the trafficking of the cell surface TCR to lysosomes To analyze how LAPTM5 affects the subcellular localization of CD3z, we retrovirally transduced the T-cell hybridoma MA5.810, which lacks endogenous CD3z expression, with CD3z. We then further transduced these cells with either pMX-LAPTM5-IRES-hCD8 or pMXIRES-hCD8 as a control, and sorted CD8 þ cells. In MA5.8 cells reconstituted with CD3z and a control vector, CD3z appeared predominantly on or near the plasma membrane, and only a small fraction colocalized with the lysosome marker LAMP-1 (Figure 1a, upper panels). By contrast, in MA5.8 cells expressing both CD3z and LAPTM5, a large portion of CD3z was localized in the lysosomal compartment (Figure 1a, lower panels). ImageJ analysis revealed a great increase of CD3z and LAMP-1 colocalization in cells expressing LAPTM5 (Figure 1b). These results suggest that overexpression of LAPTM5 alone is sufficient to promote the transport of CD3z to the lysosome. To explore whether the CD3z being transported to the lysosome by LAPTM5 was intracellular or on the cell surface associated with CD3e, CD3g, CD3d and TCRab, we next analyzed the trafficking of the cell surface TCR complex to the lysosome. We labeled the cell surface CD3e with the biotinylated agonistic 2C11 anti-CD3e antibody and Alexa Fluor 555-conjugated streptavidin (Molecular probes, Eugene, OR, USA), cultured the cells for various time periods and then analyzed them for CD3e localization. Treatment with the 2C11 anti-CD3e antibody is known to activate the TCR; therefore, the observed trafficking of the labeled CD3e reflects a TCR signaling-dependent phenomenon. As shown in Figure 1c, a fraction of the CD3e was transported to lysosomes in MA5.8 cells reconstituted with CD3z alone (upper panels), and LAPTM5 overexpression did not further promote CD3e translocation to lysosomes (lower panels). ImageJ analysis revealed similar kinetics of CD3e and LAMP-1 colocalization regardless of LAPTM5 expression (Figure 1d). Immunology and Cell Biology
As cell surface CD3z is associated with CD3e and other TCR components, these observations suggest that the lysosomal translocation of cell surface CD3z is unaffected by LAPTM5 overexpression. Immunoblot analysis of the total lysate of MA5.8 cells expressing CD3z alone (Ctrl) or CD3z þ LAPTM5 revealed that the total amount of cellular (intracellular þ cell surface) CD3z was specifically reduced by LAPTM5 (Figure 1e, input; from 57.7 to 38.5). However, antiCD3e or anti-TCRb precipitated similar amounts of CD3z from MA5.8 cells expressing CD3z alone or CD3z þ LAPTM5 (Figure 1c, CD3e IP and TCRb IP). These biochemical data are also consistent with the idea that LAPTM5 does not target CD3z already incorporated into the TCR complex. LAPTM5 targets CD3z in the Golgi apparatus Having found that LAPTM5 promoted the transport of intracellular CD3z to the lysosome, we next sought to identify the intracellular compartment where LAPTM5 targets CD3z. For this purpose, we examined the trafficking of the newly synthesized CD3z in the presence or absence of LAPTM5 overexpression. We transduced retrovirus expressing CD3z into MA5.8 cells stably expressing LAPTM5 or not, and analyzed CD3z subcellular localization at different time points. As shown in Figure 2a, CD3z was already expressed at 7 h after virus transduction. Confocal microscopic analysis revealed that the newly synthesized CD3z did not yet colocalize with either the Golgi marker Golgin or the lysosome marker LAMP-1 (Figure 2a, upper panels), suggesting that these CD3z chains likely reside at ER. At this time point, LAPTM5 expression did not result in the lysosomal translocation of CD3z (Figure 2a, lower panels), suggesting that LAPTM5 did not target CD3z localized in the ER. At 9 h after virus transduction, CD3z in control cells had apparently reached the Golgi but was not transported to lysosomes (Figure 2b, upper panels). By contrast, in cells expressing LAPTM5, a fraction of the CD3z had been transported to the lysosomal compartment (Figure 2b, lower panels). The lysosomal translocation of CD3z was even more evident in cells expressing LAPTM5 at 12 h after virus transduction (Figure 2c, lower panels). ImageJ analysis confirmed the increased CD3z and LAMP-1 colocalization after 9 h of CD3z virus transduction in cells expressing LAPTM5 relative to control cells (Figure 2d). Concomitantly, the colocalization of CD3z and Golgin was decreased in cells expressing LAPTM5 as compared with control cells (Figure 2e), likely due to the enhanced lysosomal translocation of CD3z by LAPTM5. Collectively, these results suggest that LAPTM5 targets CD3z after it leaves the ER and reaches the Golgi apparatus and promotes its lysosomal translocation for degradation. In agreement with this possibility, we found that LAPTM5-mediated CD3z degradation was partially inhibited by Brefeldin A, which is known to inhibit the export of newly synthesized proteins from ER to the Golgi (Figure 2f). Furthermore, these results also indicate that LAPTM5 unlikely targets CD3z at ER, in which case Brefeldin A would be expected to enhance CD3z degradation. A Golgi-localizing CD3z mutant is efficiently targeted by LAPTM5 To further verify whether the Golgi is the site where LAPTM5 targets CD3z, we attached the golgin-97, RanBP2a, Imh1p and p230/golgin245 (GRIP) domain of the Golgi-localizing protein Golgin to the C terminus of CD3z. As expected, the CD3z–GRIP fusion protein predominantly localized in the Golgi in both MA5.8 (Figure 3a, lower panels) and TG40 T cells,28 which lack endogenous TCRab expression (Figure 3b, lower panels). If LAPTM5 really targets CD3z in the Golgi apparatus, the CD3z–GRIP should be sensitive to LAPTM5-mediated
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Figure 1 LAPTM5 promotes the lysosomal translocation of intracellular but not of cell surface CD3z. (a) MA5.8 cells were transduced with retrovirus expressing pMX-CD3z-IRES-rCD2, and rCD2 þ cells were sorted. The sorted cells were further transduced with retrovirus expressing pMX-LAPTM5-IREShCD8 or pMX-IRES-hCD8 as a control, and CD8 þ cells were sorted. The cells expressing exogenous CD3z with or without LAPTM5 were incubated for 6 h in the presence of 20 mM NH4Cl (lysosome inhibitor), fixed, permeabilized, blocked and immunostained with anti-CD3z together with anti-LAMP-1 (lysosome marker) antibodies. Representative results of five independent experiments are shown. Arrowheads show colocalization of CD3z and LAMP-1. Scale bars, 3 mm. (b) Images of CD3z and LAMP-1 in over 100 control and LAPTM5-expressing cells were analyzed with WCIF ImageJ software, and mean±s.d. of Pearson’s coefficient is presented as the extent of colocalization between CD3z and LAMP-1. (c) Subcellular trafficking of cell surface CD3e in MA5.8 cells expressing exogenous CD3z with or without LAPTM5. Cell surface CD3e was bound with biotinylated anti-CD3e antibody plus Alexa Fluor 555-conjugated streptavidin, washed, cultured in medium for various periods, fixed, permeabilized, blocked and immunostained with anti-LAMP-1 Ab. The results 20 min after culture are shown and arrowheads indicate labeled CD3e transported to the lysosomes. Representative results of three independent experiments are shown. Scale bars, 3 mm. (d) Images of CD3e and LAMP-1 in over 100 control and LAPTM5-expressing cells at each time point are compared with WCIF ImageJ software, and mean±s.d. of Pearson’s coefficient is presented as the extent of colocalization between labeled CD3e and the lysosome. (e) CD3z and CD3e protein levels in total lysate (Input), or after immunoprecipitation with anti-CD3e or anti-TCRb. Data are representative of three independent experiments. Shown below each band is the band intensity (mean±s.d.) quantitated with a Luminescent Image Analyzer (LAS-3000 mini, Fuji Film, Tokyo, Japan).
degradation. We therefore compared the efficiency of LAPTM5mediated degradation of WT CD3z and CD3z–GRIP in MA5.8 and TG40 cells. As shown in Figure 3c and d, CD3z–GRIP was degraded by LAPTM5 in both MA5.8 and TG40 cells, although the degradation efficiency in MA5.8 cells was lower compared with WT CD3z. Confocal microscopic analysis confirmed that LAPTM5 promoted the transport of the Golgi-localizing CD3z–GRIP to the lysosomal compartment (Figure 3e). Although the expression levels and the degradation efficiency of CD3z–GRIP somewhat differed in MA5.8 and TG40 T cells, the fact that CD3z–GRIP could be degraded in both cells is consistent with the finding that the Golgi is the site where LAPTM5 targets CD3z and transports it to the lysosomal compartment.
The degradation of the Golg-localizing CD3z–GRIP by LAPTM5 further suggests that LAPTM5-mediated CD3z degradation is independent of TCR signaling. The tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) of CD3z are known to be phosphorylated upon ligand binding to TCR. To verify that tyrosine phosphorylation of CD3z is not required for LAPTM5mediated degradation, we mutated all six tyrosine residues in the three ITAMs to phenylalanines (YF mutant) and analyzed sensitivity to LAPTM5. As shown in Figure 3f, the CD3z YF mutant was degraded as efficiently as was WT CD3z. These results suggest that LAPTM5-mediated CD3z degradation is independent of ITAM phosphorylation induced by TCR signaling, in agreement with the Immunology and Cell Biology
LAPTM5 targets intracellular CD3z Y Kawai et al 530
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CD3ζ Figure 2 LAPTM5 targets CD3z in the Golgi apparatus and promotes its lysosomal translocation. (a–c) Kinetic analysis of the subcellular localization of the newly synthesized CD3z in MA5.8 cells expressing or not expressing LAPTM5. MA5.8 cells were transduced with retrovirus expressing pMX-LAPTM5-IREShCD8 or pMX-IRES-hCD8 as a control, and the hCD8 þ cells were sorted. The sorted cells were further transduced with retrovirus expressing pMX- CD3zIRES-rCD2 and cultured for 7 h (a), 9 h (b) or 12 h (c) in the presence of 20 mM NH4Cl for the last 3 h, fixed, permeabilized, blocked and immunostained with anti-CD3z, anti-Golgin and anti-LAMP-1 antibodies. Representative images of three independent experiments are shown. The extent of colocalization of CD3z with LAMP-1 (d) or with Golgin (e) was analyzed by ImageJ software. Over 50 control and LAPTM5-expressing cells at each time point were compared and mean±s.d. of Pearson’s coefficient is presented. *Po0.05 (unpaired t-test). (f) LAPTM5-mediated CD3z degradation was partially inhibited by Brefeldin A, which inhibits the export of newly synthesized proteins from ER to the Golgi. MA5.8 cells expressing CD3z with or without LAPTM5 expression were left untreated or treated with Brefeldin A for 6 h and analyzed for the cellular CD3z levels by intracellular staining. Representative FACS profiles are shown and mean±s.d. of three independent experiments is indicated.
observation that LAPTM5 targets intracellular CD3z but not surface CD3z associated with the mature TCR complex. LAPTM5 and SLAP target CD3z through distinct genetic pathways Earlier studies have revealed that SLAP, an adapter protein specifically expressed in thymic CD4 and CD8 DP cells, is involved in the lysosomal translocation and degradation of CD3z internalized from the cell surface together with other TCR subunits. Consequently, DP cells in SLAP-deficient mice express higher levels of cell surface TCR than do WT DP cells. Our data presented thus far suggest that LAPTM5 promotes the lysosomal translocation of intracellular but not cell surface CD3z, suggesting that LAPTM5 and SLAP function in distinct pathways to mediate CD3z degradation. Consistent with this notion, we found that TCR levels were further elevated on DP cells of SLAP/LAPTM5 double KO (DKO) mice as compared with the SLAP or LAPTM5 singly deficient DP cells (Figures 4a and b). These mice also expressed a transgenic TCR (DO11.10) recognizing the OVA peptide presented by major histocompatibility complex class II Immunology and Cell Biology
molecule, and their DP thymocytes were positively selected to differentiate into CD4 single-positive cells on the H-2d selecting background.29 We found that positive selection of the OVA-specific CD4 þ T cells was further enhanced in DKO mice (Figure 4c). It is interesting to note that TCR levels were only marginally increased in LAPTM5 KO cells compared with WT cells (Figure 4b), whereas TCR levels were more clearly elevated in DKO cells compared with SLAP KO cells (Figure 4b). These observations strongly suggest that LAPTM5 and SLAP function in distinct genetic pathways to mediate CD3z degradation and negatively regulate TCR expression.
DISCUSSION Tight regulation of cell surface TCR expression is critical for normal T-cell development and optimal activation. In the present study we show that the lysosomal protein LAPTM5 targets intracellular CD3z in the Golgi and promotes its lysosomal translocation for degradation while having little affect on the cell surface CD3z associated with the
LAPTM5 targets intracellular CD3z Y Kawai et al 531
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Figure 3 A Golgi-localizing CD3z–GRIP fusion protein can be transported to the lysosome and degraded by LAPTM5. (a, b) Subcellular localization of WT CD3z or CD3z–GRIP chimera in MA5.8 (a) or TG40 cells (b). MA5.8 or TG40 cells were transduced with retrovirus expressing pMX-CD3z-IRES-rCD2 or pMX-CD3z-GRIP-IRES-rCD2. The rCD2 þ cells were sorted and stained with anti-CD3z and Golgin. Arrowheads indicate colocalization of CD3z–GRIP with Golgin. Data are representative of three independent experiments. (c, d) LAPTM5-mediated degradation of WT CD3z and CD3z–GRIP in MA5.8 (c) and TG40 (d) cells. MA5.8 (c) or TG40 cells (d) expressing WT CD3z or CD3z–GRIP were transduced with retrovirus expressing pMX-LAPTM5-IRES-hCD8 or pMX-IRES-hCD8 as a control and analyzed for cellular CD3z expression by intracellular staining with an anti-CD3z antibody. Data are representative of four independent experiments and mean±s.d. is shown. (e) Subcellular localization of CD3z–GRIP in control TG40 or TG40 cells expressing LAPTM5. Representative images are shown. Arrowheads indicate colocalization of CD3z–GRIP and LAMP-1. Scale bars, 3 mm. (f) The CD3z YF mutant is equally sensitive to LAPTM5-mediated degradation. MA5.8 cells expressing pMX-CD3z-IRES-rCD2 or pMX-CD3zYF-IRES-rCD2 were transduced with retrovirus expressing pMX-LAPTM5-IRES-hCD8 or pMX-IRES-hCD8 as a control and analyzed for intracellular CD3z levels after gating on hCD8 þ cells. Representative FACS profiles are shown and mean±s.d. of three independent experiments is indicated.
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Figure 4 LAPTM5 and SLAP mediate TCR degradation through distinct genetic pathways. (a) Cell surface TCR levels on thymic DP cells from WT, LAPTM5 KO, SLAP KO, and LAPTM5 and SLAP DKO DO11.10 TCR transgenic mice (6-8-week-old age-matched). Data are representative of three independent experiments. (b) The results of three mice of each genotype are depicted. (c) Enhanced positive selection of DP thymocytes in LAPTM5 and SLAP DKO mice. Percentages of CD4 þ CD8 thymocytes of three mice of each genotype are depicted. A horizontal bar indicates the mean. *Po0.05; **Po0.01; ***Po0.001 (one-way analysis of variance þ post hoc test). MFI, mean fluorescence intensity.
TCR complex. Consistent with this finding, we show that the tyrosine phosphorylation of CD3z ITAMs is not required for LAPTM5mediated CD3z degradation. These results suggest that CD3z can
be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3z dependent on TCR signaling, and LAPTM5, which targets intracellular CD3z independent of TCR signaling. Immunology and Cell Biology
LAPTM5 targets intracellular CD3z Y Kawai et al 532
Our findings provide new insights into our understanding of the precise regulation of TCR expression and T-cell activation. We initially found that LAPTM5 promoted the lysosomal translocation of intracellular CD3z but not of cell surface CD3z. Kinetic analysis further revealed that the Golgi appears to be the site where LAPTM5 targets CD3z. It is interesting to note that LAPTM5 itself has been shown to localize in the Golgi before being sorted to the lysosome where it normally resides.30,31 These observations raise the possibility that LAPTM5 promotes the lysosomal translocation of CD3z during its own trafficking from the Golgi to lysosomes. Recently, LAPTM5 has been suggested to transport the mH chain in the pre-BCR complex from the ER to lysosomes.32 It remains to be determined whether the pre-BCR is transported to the Golgi before being sorted to lysosomes. Proteins transported from the ER enter the cis-Golgi network and then exit at the trans-Golgi network, which is a particularly important sorting station among Golgi stacks and directs newly synthesized proteins to different subcellular destinations.33 We speculate that the trans-Golgi network is the discrete site where LAPTM5 functions because the GRIP domain has been suggested to promote targeting of Golgin to the trans-Golgi network34 and the CD3z–GRIP fusion protein could be degraded by LAPTM5. It is possible that LAPTM5 promotes the transport of CD3z into lysosomes by altering its sorting process in the trans-Golgi network. The ITAM is defined by the sequence Yxx(L/I)x6-8Yxx(L/I), and the Y-residues are particularly emblematic as they are phosphorylated and form docking sites for signaling molecules on immunoreceptor triggering. However, we demonstrate that phosphorylation of tyrosines in the ITAMs is dispensable for degradation by LAPTM5 as the WT and CD3z YF mutant are comparably degraded by LAPTM5. These observations suggest that LAPTM5 functions independently of TCR signaling and are well consistent with the result that LAPTM5 preferentially targets intracellular CD3z in the Golgi. ITAM tyrosines are also known to function as an endocytic signal and to promote the internalization and trafficking of CD3z into the lysosome, possibly regardless of phosphorylation status.17 The finding that tyrosine residues of CD3z are not required for LAPTM5-mediated degradation further supports our conclusion that LAPTM5 targets intracellular but not cell surface CD3z associated with the TCR complex. We have previously shown that LAPTM5 deficiency did not affect TCR expression in naive T cells but caused elevated TCR levels after CD3 stimulation. These earlier observations suggested that LAPTM5 function was TCR dependent in primary T cells, a conclusion different from the current study. In our earlier study, primary spleen T cells stimulated with anti-CD3 antibody downmodulated surface TCR, which reached the lowest level at 12 h after stimulation (26). The TCR levels then recovered after 24 h, which correlated with the increased CD3z protein levels (26, 35). Notably, it was during this recovery period that LAPTM5 deficiency led to elevated surface TCR levels, consistent with the current data showing that LAPTM5 targets newly synthesized CD3z. The fact that LAPTM5 deficiency did not affect TCR levels in unstimulated T cells could be due to low levels of TCR metabolism (internalization, recycling and new synthesis) in these cells. Therefore, we think that LAPTM5 preferentially targets newly synthesized CD3z and its function in primary T cells becomes more evident after TCR stimulation, which causes dynamic TCR metabolism and resynthesis of a new TCR complex. Our results, however, do not exclude the possibility that LAPTM5 may also target the internalized TCR complex under certain circumstances in primary cells. Earlier studies have revealed that the adapter protein SLAP and the E3 ubiquitin ligase c-Cbl together regulate TCR levels by targeting the Immunology and Cell Biology
CD3z in the internalized TCR complex for ubiquitination and subsequent lysosomal degradation. The present study shows that LAPTM5 does not affect the fate of the internalized TCR complex but instead targets intracellular CD3z and promotes its lysosomal translocation for degradation. In other words, SLAP/c-Cbl regulates the fate of the fully assembled surface TCR, whereas LAPTM5 regulates the amount of the newly synthesized TCR. Therefore, LAPTM5 and SLAP/c-Cbl mediate CD3z degradation through distinct pathways. This conclusion is further supported by genetic data showing that LAPTM5 and SLAP DKO thymocytes express higher levels of cellular CD3z and surface TCR compared with singly deficient thymocytes. The DKO mice also show enhanced positive selection of the DP thymocytes, suggesting that the elevated TCR levels indeed result in increased TCR signaling required for efficient positive selection and differentiation into the more mature singlepositive thymocytes. It has been shown that CD3z protein levels decrease first and then increase at late time points after TCR signaling.35 The time course of CD3z expression correlates with the cell surface TCR levels. Notably, LAPTM5 protein levels also increase at late time points after T-cell activation, presumably to prevent overshooting of normal TCR expression levels, which would lead to excessive and/or prolonged T-cell activation. In fact, our earlier studies have revealed that TCR levels on LAPTM5 KO spleen T cells become much higher than those on WT spleen T cells only at late time points (24 h and 48 h) after TCR crosslinking.26 Collectively, the LAPTM5 pathway functions at a relatively late phase when T cells start to recover from their downmodulated TCR expression by assembly of newly synthesized TCR subunits, in contrast to the SLAP/c-Cbl pathway that functions during an early phase of TCR signaling after TCR internalization. Therefore, T cells likely possess at least two layers of regulatory mechanisms that function at different time points and in distinct cellular locations to strictly control surface TCR expression for normal T-cell development and activation. The present study has revealed a new pathway that promotes CD3z degradation. It has been reported that abnormal TCR lacking some of the TCR subunits could reach and reside on the cell surface to some extent, partially overcoming the developmental block at the positive selection stage, as was observed in CD3g-, d- and z-deficient mice.36–38 In addition, earlier studies demonstrated that high-level expression of human CD3d rescued TCR expression in CD3g- and CD3d-deficient mice,39 raising the possibility that some TCR subunits, if produced in excess, could be substituted for deficient subunits in the formation of an abnormal TCR complex. Cell surface expression of the partially assembled receptor in the absence of one of the receptor components may result in the generation of autoreactive T cells.40,41 In this regard, it will be interesting to determine whether LAPTM5 suppresses abnormal TCR expression by degrading excessive intracellular TCR subunits during TCR assembly. METHODS Mice LAPTM5 KO, SLAP KO and DO11.10 TCR transgenic mice have been described previously.24,26 All the mice were maintained under specific pathogen-free conditions and all experiments were approved by the Animal Facility Committee of RIKEN Yokohama Institute (Permission number 20-025).
Antibodies and other reagents The following antibodies were used: anti-LAPTM5,26 anti-LAMP-1 (1D4B, BD Biosciences, San Jose, CA, USA) and anti-Golgin-97 (CDF4, Molecular Probes) for immunostaining; anti-TCRb (H57-597, Biolegend, San Diego, CA,
LAPTM5 targets intracellular CD3z Y Kawai et al 533 USA), anti-CD3e (145-2C11, Biolegend), anti-CD4 (GK1.5, Biolegend), antiCD8 (Ly-2, Biolegend), anti-rat CD2 (OX-34, BD Biosciences) and antihuman CD8 (RPA-T8, eBioscience, San Diego, CA, USA) for flow cytometry; anti-ACTIN (rabbit polyclonal antibodies, Sigma, St Louis, MO, USA) and anti-CD3e (M-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for western blotting; anti-CD3z mAb (H146) used in all applications in this study was a kind gift from Dr T. Saito (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan).
Cells and culture Both MA5.8 and TG40 are variants of the T-cell hybridoma 2B4.11 specific for I-Ek-restricted cytochrome c peptide.10,28 These cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, 50 mM 2-mercaptoethanol and antibiotics. The PHOENIX packaging cell line was maintained in Dulbecco’s modified Eagle medium medium supplemented with 10% fetal calf serum and antibiotics.
Construction and retroviral transduction The CD3z–GRIP chimera consists of full-length CD3z C-terminally fused to 100 amino acids of Golgin-97 (659–759). YF mutant of CD3z, in which all the six Tyr in three ITAMs are mutated to Phe, was generated by site-directed mutagenesis using the PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc., Shiga, Japan). DNA fragments encoding these proteins including WT CD3z and LAPTM5 were inserted into the EcoRI/XhoI site of pMX-IRES-rCD2, pMX-IRES-hCD8 or pMX-IRES-GFP retroviral vector. The retroviral constructs were transfected into the PHOENIX packaging cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and the viral supernatant was prepared as described previously.26 Virus transduction was carried out in the presence of 10 mg ml 1 DOTAP (Roche, Basel, Switzerland). In experiments analyzing the effect of LAPTM5 on CD3z/CD3e localization and degradation, MA5.8 or TG40 cells were transduced with retrovirus expressing either LAPTM5 or the transduction marker (rCD2, hCD8 or GFP) alone as a control and cells with similar levels of the transduction marker were sorted. Appropriate combinations of retroviruses were used in transduction of MA5.8 or TG40 cells as indicated in the figure legend.
Immunostaining Cells were fixed with 6% formalin/phosphate-buffered saline (PBS) for 15 min at room temperature, permeabilized with 0.1% Triton-X-100/PBS for 5 min, washed, blocked with 1% bovine serum albumin/PBS and incubated with primary antibodies at room temperature for 1 h followed by the Alexa Fluor 488-, 555- or 647-conjugated secondary antibodies for 30 min. The cells were examined under a confocal microscope (SP2AOBS, Leica Microsysytems, Wetzlar, Germany), and photographs were recorded with Leica confocal software.
Flow cytometry Multicolor flow cytometric analysis was performed with FACSCalibur (BD Biosciences). Analysis of intracellular molecules was performed as follows: cells were fixed with 6% formalin/PBS for 10 min at 37 1C, permeabilized with 0.2% Tween 20/PBS for 20 min, washed, blocked with 1% bovine serum albumin/ PBS and incubated with staining antibodies at room temperature for 20 min. The cellular CD3z was stained with H146 antibody followed by PE-conjugated anti-hamster IgG.
Immunoprecipitation and immunoblotting Cells were lysed at 4 1C for 10 min in lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA and complete protease inhibitor cocktail tablets (Roche) containing 1% NP-40) and were centrifuged at 20 000 g for 5 min, following which the supernatant was recovered. The lysates were incubated with indicated biotinylated antibodies at 4 1C for 2 h, followed by incubation with streptavidin sepharose (GE Healthcare, Little Chalfont, UK) at 4 1C for 30 min, and the immunoprecipitates were washed four times with lysis buffer, resuspended with sample buffer and boiled for 5 min. The samples were resolved by PAGE, transferred to immobilon-P membranes
(Millipore, Billerica, MA, USA), blocked with blocking one (Nacalai, Kyoto, Japan), incubated with the indicated primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, and then developed with Immobilon Western Chemiluminescent horseradish peroxidase substrate (Millipore).
Statistical analysis Statistical significance was assessed with the unpaired Student t-test for Figure 2 and analysis of variance þ post hoc test for Figure 4. *Po0.05; **Po0.01; ***Po0.001.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We are grateful to Drs T. Saito and T. Yokosuka for providing the MA5.8 and TG40 cells and the anti-CD3z mAb. We also thank Drs A. Weiss, S. Fagarasan, H. Kawamoto, K. Masuda, K. Kakugawa and K. Kometani for helpful suggestions, the Animal Facility for maintaining the mice and the FACS Facility for cell sorting.
1 Kuhns MS, Davis MM, Garcia KC. Deconstructing the form and function of the TCR/CD3 complex. Immunity 2006; 24: 133–139. 2 Viola A, Lanzavecchia A. T cell activation determined by T cell receptor number and tunable thresholds. Science 1996; 273: 104–106. 3 Liu H, Rhodes M, Wiest DL, Vignali DA. On the dynamics of TCR:CD3 complex cell surface expression and downmodulation. Immunity 2000; 13: 665–675. 4 Geisler C. TCR trafficking in resting and stimulated T cells. Crit Rev Immunol 2004; 24: 67–86. 5 Alarcon B, Berkhout B, Breitmeyer J, Terhorst C. Assembly of the human T cell receptor-CD3 complex takes place in the endoplasmic reticulum and involves intermediary complexes between the CD3-gamma.delta.epsilon core and single T cell receptor alpha or beta chains. J Biol Chem 1988; 263: 2953–2961. 6 Koning F, Maloy WL, Coligan JE. The implications of subunit interactions for the structure of the T cell receptor-CD3 complex. Eur J Immunol 1990; 20: 299–305. 7 Kearse KP, Roberts JL, Singer A. TCR alpha-CD3 delta epsilon association is the initial step in alpha beta dimer formation in murine T cells and is limiting in immature CD4 þ CD8 þ thymocytes. Immunity 1995; 2: 391–399. 8 Dietrich J, Kastrup J, Lauritsen JP, Menne C, von Bulow F, Geisler C. TCRzeta is transported to and retained in the Golgi apparatus independently of other TCR chains: implications for TCR assembly. Eur J Immunol 1999; 29: 1719–1728. 9 Weissman AM, Frank SJ, Orloff DG, Mercep M, Ashwell JD, Klausner RD. Role of the zeta chain in the expression of the T cell antigen receptor: genetic reconstitution studies. EMBO J 1989; 8: 3651–3656. 10 Sussman JJ, Bonifacino JS, Lippincott-Schwartz J, Weissman AM, Saito T, Klausner RD et al. Failure to synthesize the T cell CD3-zeta chain: structure and function of a partial T cell receptor complex. Cell 1988; 52: 85–95. 11 Delgado P, Alarcon B. An orderly inactivation of intracellular retention signals controls surface expression of the T cell antigen receptor. J Exp Med 2005; 201: 555–566. 12 Lee SJ. Endoplasmic reticulum retention and degradation of T cell antigen receptor beta chain. Exp Mol Med 1998; 30: 159–164. 13 Bonifacino JS, Suzuki CK, Klausner RD. A peptide sequence confers retention and rapid degradation in the endoplasmic reticulum. Science 1990; 247: 79–82. 14 Mallabiabarrena A, Fresno M, Alarcon B. An endoplasmic reticulum retention signal in the CD3 epsilon chain of the T-cell receptor. Nature 1992; 357: 593–596. 15 Mallabiabarrena A, Jimenez MA, Rico M, Alarcon B. A tyrosine-containing motif mediates ER retention of CD3-epsilon and adopts a helix-turn structure. EMBO J 1995; 14: 2257–2268. 16 Letourneur F, Klausner RD. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell 1992; 69: 1143–1157. 17 Bonifacino JS, Traub LM. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem 2003; 72: 395–447. 18 Huppa JB, Ploegh HL. The alpha chain of the T cell antigen receptor is degraded in the cytosol. Immunity 1997; 7: 113–122. 19 Letourneur F, Klausner RD. Activation of T cells by a tyrosine kinase activation domain in the cytoplasmic tail of CD3 epsilon. Science 1992; 255: 79–82. 20 Lauritsen JP, Bonefeld CM, von Essen M, Nielsen MW, Rasmussen AB, Odum N et al. Masking of the CD3 gamma di-leucine-based motif by zeta is required for efficient T-cell receptor expression. Traffic 2004; 5: 672–684. 21 Wiest DL, Yuan L, Jefferson J, Benveniste P, Tsokos M, Klausner RD et al. Regulation of T cell receptor expression in immature CD4 þ CD8 þ thymocytes by p56lck tyrosine kinase: basis for differential signaling by CD4 and CD8 in immature thymocytes expressing both coreceptor molecules. J Exp Med 1993; 178: 1701–1712.
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LAPTM5 targets intracellular CD3z Y Kawai et al 534 22 Cosgrove D, Gray D, Dierich A, Kaufman J, Lemeur M, Benoist C et al. Mice lacking MHC class II molecules. Cell 1991; 66: 1051–1066. 23 Molina TJ, Kishihara K, Siderovski DP, van Ewijk W, Narendran A, Timms E et al. Profound block in thymocyte development in mice lacking p56lck. Nature 1992; 357: 161–164. 24 Myers MD, Dragone LL, Weiss A. Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCRzeta for degradation. J Cell Biol 2005; 170: 285–294. 25 Myers MD, Sosinowski T, Dragone LL, White C, Band H, Gu H et al. Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex. Nat Immunol 2006; 7: 57–66. 26 Ouchida R, Yamasaki S, Hikida M, Masuda K, Kawamura K, Wada A et al. A lysosomal protein negatively regulates surface T cell antigen receptor expression by promoting CD3zeta-chain degradation. Immunity 2008; 29: 33–43. 27 Ouchida R, Kurosaki T, Wang JY. A role for lysosomal-associated protein transmembrane 5 in the negative regulation of surface B cell receptor levels and B cell activation. J Immunol 2010; 185: 294–301. 28 Ohno H, Ushiyama C, Taniguchi M, Germain RN, Saito T. CD2 can mediate TCR/CD3independent T cell activation. J Immunol 1991; 146: 3742–3746. 29 Sosinowski T, Killeen N, Weiss A. The Src-like adaptor protein downregulates the T cell receptor on CD4 þ CD8 þ thymocytes and regulates positive selection. Immunity 2001; 15: 457–466. 30 Pak Y, Glowacka WK, Bruce MC, Pham N, Rotin D. Transport of LAPTM5 to lysosomes requires association with the ubiquitin ligase Nedd4, but not LAPTM5 ubiquitination. J Cell Biol 2006; 175: 631–645. 31 Ishihara T, Inoue J, Kozaki K, Imoto I, Inazawa J. HECT-type ubiquitin ligase ITCH targets lysosomal-associated protein multispanning transmembrane 5 (LAPTM5) and prevents LAPTM5-mediated cell death. J Biol Chem 2011; 286: 44086–44094. 32 Kawano Y, Ouchida R, Wang JY, Yoshikawa S, Yamamoto M, Kitamura D et al. A novel mechanism for the autonomous termination of pre-B cell receptor expression via
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induction of lysosome-associated protein transmembrane 5. Mol Cell Biol 2012; 32: 4462–4471. 33 Gu F, Crump CM, Thomas G. Trans-Golgi network sorting. Cell Mol Life Sci 2001; 58: 1067–1084. 34 Man Z, Kondo Y, Koga H, Umino H, Nakayama K, Shin HW. Arfaptins are localized to the trans-Golgi by interaction with Arl1, but not Arfs. J Biol Chem 2011; 286: 11569–11578. 35 Bronstein-Sitton N, Wang L, Cohen L, Baniyash M. Expression of the T cell antigen receptor zeta chain following activation is controlled at distinct checkpoints. Implications for cell surface receptor down-modulation and re-expression. J Biol Chem 1999; 274: 23659–23665. 36 Haks MC, Krimpenfort P, Borst J, Kruisbeek AM. The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages. EMBO J 1998; 17: 1871–1882. 37 Dave VP, Cao Z, Browne C, Alarcon B, Fernandez-Miguel G, Lafaille J et al. CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage. EMBO J 1997; 16: 1360–1370. 38 Love PE, Shores EW, Johnson MD, Tremblay ML, Lee EJ, Grinberg A et al. T cell development in mice that lack the zeta chain of the T cell antigen receptor complex. Science 1993; 261: 918–921. 39 Fernandez-Malave E, Wang N, Pulgar M, Schamel WW, Alarcon B, Terhorst C. Overlapping functions of human CD3delta and mouse CD3gamma in alphabeta T-cell development revealed in a humanized CD3gamma-mouse. Blood 2006; 108: 3420–3427. 40 Lin SY, Ardouin L, Gillet A, Malissen M, Malissen B. The single positive T cells found in CD3-zeta/eta-/- mice overtly react with self-major histocompatibility complex molecules upon restoration of normal surface density of T cell receptor-CD3 complex. J Exp Med 1997; 185: 707–715. 41 Yamazaki T, Arase H, Ono S, Ohno H, Watanabe H, Saito T. A shift from negative to positive selection of autoreactive T cells by the reduced level of TCR signal in TCR-transgenic CD3 zeta-deficient mice. J Immunol 1997; 158: 1634–1640.
ORIGINAL ARTICLE
LAPTM5 promotes lysosomal degradation of intracellular CD3f but not of cell surface CD3f Yohei Kawai1, Rika Ouchida1, Sho Yamasaki2, Leonard Dragone3, Takeshi Tsubata5 and Ji-Yang Wang4,5 The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3f degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3f but not of the cell surface CD3f associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3f suggests that LAPTM5 targets CD3f in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3f can be transported to and degraded in the lysosome by LAPTM5. A CD3f YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3f, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3f is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3f in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3f can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3f dependent on TCR signaling, and LAPTM5, which targets intracellular CD3f independent of TCR signaling. Immunology and Cell Biology (2014) 92, 527–534; doi:10.1038/icb.2014.18; published online 18 March 2014
T lymphocytes, which have a central role in immune regulation, recognize processed antigens in the context of major histocompatibility complex by virtue of their T cell receptor (TCR). The TCR is a multi-subunit complex composed of the variable antigenbinding TCRab or gd chains that are associated with the invariant signal-transducing CD3ge, de- and zz-subunits.1 TCR signaling must be strictly controlled to allow for normal T-cell development and optimal activation. One important mechanism for regulating the TCR signal is the modulation of its cell surface expression.2 Cell surface TCR levels are the result of a dynamic equilibrium among at least the following four processes: the assembly of the newly synthesized TCR and its transport to the cell surface; internalization of the surface TCR; recycling of the internalized TCR back to the cell surface; and degradation of the internalized TCR. Modulation of any of these processes would affect the steady-state TCR levels.3,4 The TCR complex is initially assembled in the endoplasmic reticulum (ER), where CD3e dimerizes with either CD3g or CD3d and then these dimers associate with TCRa and b-chains.5–7 It has been suggested that only when all chains other than CD3z are assembled is the resulting ade–bge complex exported to the Golgi apparatus, where the CD3z homodimer is bound to form the fully assembled TCR complex.8–10 This stepwise assembly and transport are
known to be regulated by ER retention motifs11–14 and/or endocytic recognition sequences containing di-leucine or tyrosine.15–17 These functional ER retention and endocytic recognition motifs are present in all CD3 subunits other than CD3z, and all of them must be masked by association with other subunits before transport to the plasma membrane.11 The unassembled TCR subunits in the ER are rapidly degraded in the proteasome by an ER-to-cytosol transport mechanism.18 Partially assembled TCR in the Golgi, on the other hand, is degraded in lysosomes unless association of the CD3z homodimer masks di-leucine- and/or tyrosine-based motifs in CD3g, d- and e-chains.16,19,20 After transport to the cell surface, levels of the fully assembled TCR are also dynamically controlled. In CD4 þ CD8 þ double-positive (DP) thymocytes, major histocompatibility complex class II-CD4 interaction and the resultant activation of Lck, a Src family tyrosine kinase, have an important role in maintaining a 10-fold lower expression of TCR compared with peripheral T cells.21–23 Lckmediated phosphorylation of CD3 chains results in TCR endocytosis, recruitment of the Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl, and ubiquitination of CD3z and its subsequent lysosomal degradation.24,25 Accordingly, both SLAP- and c-Cbl-knockout (KO) mice exhibited higher TCR levels on DP
1Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan; 2Division of Molecular Immunology, Research Center for Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 3National Jewish Health Colorado Children’s Hospital, University of Colorado Denver, School of Medicine, Denver, CO, USA; 4Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, China and 5Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Correspondence: J-Y Wang, Department of Immunology, Shanghai Medical College, Fudan University, 130 Dong’an Road, Shanghai 200032, China. E-mail: [email protected] Received 24 November 2013; revised 4 February 2014; accepted 17 February 2014; published online 18 March 2014
LAPTM5 targets intracellular CD3z Y Kawai et al 528
thymocytes due to decreased degradation of the internalized TCR in the lysosome and its increased recycling back to the cell surface.24,25 Previously we have reported that the lysosomal protein LAPTM5 negatively regulates TCR expression by interacting with CD3z and promoting its lysosomal degradation.26 Compared with wild-type (WT) mice, LAPTM5 KO mice show elevated TCR levels on DP thymocytes and antigen-stimulated peripheral T cells. Consequently, T-cell activation is enhanced, with elevated recall responses and prolonged delayed-type hypersensitivity reactions. Furthermore LAPTM5-deficient B cells express higher levels of the BCR compared with WT B cells after antigen stimulation, and the mutant mice show elevated antibody production to foreign antigens and also produce autoantibodies as they age. Therefore, LAPTM5 is a common negative regulator of TCR and BCR expression and T- and B-cell activation.27 Despite the immunological significance of LAPTM5, the molecular mechanism by which it mediates TCR and BCR degradation remains largely unknown. In the present study, we have explored the mechanisms of LAPTM5-mediated CD3z degradation in T cells. By using T cells lacking CD3z and reconstituted with exogenous CD3z, we show that LAPTM5 targets CD3z in the Golgi and promotes its lysosomal translocation, while having little effect on the cell surface CD3z associated with the TCR complex. We also provide genetic evidence that LAPTM5 promotes CD3z degradation through a pathway distinct from SLAP/c-Cbl. These results reveal a previously unidentified mechanism that controls TCR levels. RESULTS LAPTM5 promotes the lysosomal translocation of intracellular CD3f but does not affect the trafficking of the cell surface TCR to lysosomes To analyze how LAPTM5 affects the subcellular localization of CD3z, we retrovirally transduced the T-cell hybridoma MA5.810, which lacks endogenous CD3z expression, with CD3z. We then further transduced these cells with either pMX-LAPTM5-IRES-hCD8 or pMXIRES-hCD8 as a control, and sorted CD8 þ cells. In MA5.8 cells reconstituted with CD3z and a control vector, CD3z appeared predominantly on or near the plasma membrane, and only a small fraction colocalized with the lysosome marker LAMP-1 (Figure 1a, upper panels). By contrast, in MA5.8 cells expressing both CD3z and LAPTM5, a large portion of CD3z was localized in the lysosomal compartment (Figure 1a, lower panels). ImageJ analysis revealed a great increase of CD3z and LAMP-1 colocalization in cells expressing LAPTM5 (Figure 1b). These results suggest that overexpression of LAPTM5 alone is sufficient to promote the transport of CD3z to the lysosome. To explore whether the CD3z being transported to the lysosome by LAPTM5 was intracellular or on the cell surface associated with CD3e, CD3g, CD3d and TCRab, we next analyzed the trafficking of the cell surface TCR complex to the lysosome. We labeled the cell surface CD3e with the biotinylated agonistic 2C11 anti-CD3e antibody and Alexa Fluor 555-conjugated streptavidin (Molecular probes, Eugene, OR, USA), cultured the cells for various time periods and then analyzed them for CD3e localization. Treatment with the 2C11 anti-CD3e antibody is known to activate the TCR; therefore, the observed trafficking of the labeled CD3e reflects a TCR signaling-dependent phenomenon. As shown in Figure 1c, a fraction of the CD3e was transported to lysosomes in MA5.8 cells reconstituted with CD3z alone (upper panels), and LAPTM5 overexpression did not further promote CD3e translocation to lysosomes (lower panels). ImageJ analysis revealed similar kinetics of CD3e and LAMP-1 colocalization regardless of LAPTM5 expression (Figure 1d). Immunology and Cell Biology
As cell surface CD3z is associated with CD3e and other TCR components, these observations suggest that the lysosomal translocation of cell surface CD3z is unaffected by LAPTM5 overexpression. Immunoblot analysis of the total lysate of MA5.8 cells expressing CD3z alone (Ctrl) or CD3z þ LAPTM5 revealed that the total amount of cellular (intracellular þ cell surface) CD3z was specifically reduced by LAPTM5 (Figure 1e, input; from 57.7 to 38.5). However, antiCD3e or anti-TCRb precipitated similar amounts of CD3z from MA5.8 cells expressing CD3z alone or CD3z þ LAPTM5 (Figure 1c, CD3e IP and TCRb IP). These biochemical data are also consistent with the idea that LAPTM5 does not target CD3z already incorporated into the TCR complex. LAPTM5 targets CD3z in the Golgi apparatus Having found that LAPTM5 promoted the transport of intracellular CD3z to the lysosome, we next sought to identify the intracellular compartment where LAPTM5 targets CD3z. For this purpose, we examined the trafficking of the newly synthesized CD3z in the presence or absence of LAPTM5 overexpression. We transduced retrovirus expressing CD3z into MA5.8 cells stably expressing LAPTM5 or not, and analyzed CD3z subcellular localization at different time points. As shown in Figure 2a, CD3z was already expressed at 7 h after virus transduction. Confocal microscopic analysis revealed that the newly synthesized CD3z did not yet colocalize with either the Golgi marker Golgin or the lysosome marker LAMP-1 (Figure 2a, upper panels), suggesting that these CD3z chains likely reside at ER. At this time point, LAPTM5 expression did not result in the lysosomal translocation of CD3z (Figure 2a, lower panels), suggesting that LAPTM5 did not target CD3z localized in the ER. At 9 h after virus transduction, CD3z in control cells had apparently reached the Golgi but was not transported to lysosomes (Figure 2b, upper panels). By contrast, in cells expressing LAPTM5, a fraction of the CD3z had been transported to the lysosomal compartment (Figure 2b, lower panels). The lysosomal translocation of CD3z was even more evident in cells expressing LAPTM5 at 12 h after virus transduction (Figure 2c, lower panels). ImageJ analysis confirmed the increased CD3z and LAMP-1 colocalization after 9 h of CD3z virus transduction in cells expressing LAPTM5 relative to control cells (Figure 2d). Concomitantly, the colocalization of CD3z and Golgin was decreased in cells expressing LAPTM5 as compared with control cells (Figure 2e), likely due to the enhanced lysosomal translocation of CD3z by LAPTM5. Collectively, these results suggest that LAPTM5 targets CD3z after it leaves the ER and reaches the Golgi apparatus and promotes its lysosomal translocation for degradation. In agreement with this possibility, we found that LAPTM5-mediated CD3z degradation was partially inhibited by Brefeldin A, which is known to inhibit the export of newly synthesized proteins from ER to the Golgi (Figure 2f). Furthermore, these results also indicate that LAPTM5 unlikely targets CD3z at ER, in which case Brefeldin A would be expected to enhance CD3z degradation. A Golgi-localizing CD3z mutant is efficiently targeted by LAPTM5 To further verify whether the Golgi is the site where LAPTM5 targets CD3z, we attached the golgin-97, RanBP2a, Imh1p and p230/golgin245 (GRIP) domain of the Golgi-localizing protein Golgin to the C terminus of CD3z. As expected, the CD3z–GRIP fusion protein predominantly localized in the Golgi in both MA5.8 (Figure 3a, lower panels) and TG40 T cells,28 which lack endogenous TCRab expression (Figure 3b, lower panels). If LAPTM5 really targets CD3z in the Golgi apparatus, the CD3z–GRIP should be sensitive to LAPTM5-mediated
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Figure 1 LAPTM5 promotes the lysosomal translocation of intracellular but not of cell surface CD3z. (a) MA5.8 cells were transduced with retrovirus expressing pMX-CD3z-IRES-rCD2, and rCD2 þ cells were sorted. The sorted cells were further transduced with retrovirus expressing pMX-LAPTM5-IREShCD8 or pMX-IRES-hCD8 as a control, and CD8 þ cells were sorted. The cells expressing exogenous CD3z with or without LAPTM5 were incubated for 6 h in the presence of 20 mM NH4Cl (lysosome inhibitor), fixed, permeabilized, blocked and immunostained with anti-CD3z together with anti-LAMP-1 (lysosome marker) antibodies. Representative results of five independent experiments are shown. Arrowheads show colocalization of CD3z and LAMP-1. Scale bars, 3 mm. (b) Images of CD3z and LAMP-1 in over 100 control and LAPTM5-expressing cells were analyzed with WCIF ImageJ software, and mean±s.d. of Pearson’s coefficient is presented as the extent of colocalization between CD3z and LAMP-1. (c) Subcellular trafficking of cell surface CD3e in MA5.8 cells expressing exogenous CD3z with or without LAPTM5. Cell surface CD3e was bound with biotinylated anti-CD3e antibody plus Alexa Fluor 555-conjugated streptavidin, washed, cultured in medium for various periods, fixed, permeabilized, blocked and immunostained with anti-LAMP-1 Ab. The results 20 min after culture are shown and arrowheads indicate labeled CD3e transported to the lysosomes. Representative results of three independent experiments are shown. Scale bars, 3 mm. (d) Images of CD3e and LAMP-1 in over 100 control and LAPTM5-expressing cells at each time point are compared with WCIF ImageJ software, and mean±s.d. of Pearson’s coefficient is presented as the extent of colocalization between labeled CD3e and the lysosome. (e) CD3z and CD3e protein levels in total lysate (Input), or after immunoprecipitation with anti-CD3e or anti-TCRb. Data are representative of three independent experiments. Shown below each band is the band intensity (mean±s.d.) quantitated with a Luminescent Image Analyzer (LAS-3000 mini, Fuji Film, Tokyo, Japan).
degradation. We therefore compared the efficiency of LAPTM5mediated degradation of WT CD3z and CD3z–GRIP in MA5.8 and TG40 cells. As shown in Figure 3c and d, CD3z–GRIP was degraded by LAPTM5 in both MA5.8 and TG40 cells, although the degradation efficiency in MA5.8 cells was lower compared with WT CD3z. Confocal microscopic analysis confirmed that LAPTM5 promoted the transport of the Golgi-localizing CD3z–GRIP to the lysosomal compartment (Figure 3e). Although the expression levels and the degradation efficiency of CD3z–GRIP somewhat differed in MA5.8 and TG40 T cells, the fact that CD3z–GRIP could be degraded in both cells is consistent with the finding that the Golgi is the site where LAPTM5 targets CD3z and transports it to the lysosomal compartment.
The degradation of the Golg-localizing CD3z–GRIP by LAPTM5 further suggests that LAPTM5-mediated CD3z degradation is independent of TCR signaling. The tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) of CD3z are known to be phosphorylated upon ligand binding to TCR. To verify that tyrosine phosphorylation of CD3z is not required for LAPTM5mediated degradation, we mutated all six tyrosine residues in the three ITAMs to phenylalanines (YF mutant) and analyzed sensitivity to LAPTM5. As shown in Figure 3f, the CD3z YF mutant was degraded as efficiently as was WT CD3z. These results suggest that LAPTM5-mediated CD3z degradation is independent of ITAM phosphorylation induced by TCR signaling, in agreement with the Immunology and Cell Biology
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CD3ζ Figure 2 LAPTM5 targets CD3z in the Golgi apparatus and promotes its lysosomal translocation. (a–c) Kinetic analysis of the subcellular localization of the newly synthesized CD3z in MA5.8 cells expressing or not expressing LAPTM5. MA5.8 cells were transduced with retrovirus expressing pMX-LAPTM5-IREShCD8 or pMX-IRES-hCD8 as a control, and the hCD8 þ cells were sorted. The sorted cells were further transduced with retrovirus expressing pMX- CD3zIRES-rCD2 and cultured for 7 h (a), 9 h (b) or 12 h (c) in the presence of 20 mM NH4Cl for the last 3 h, fixed, permeabilized, blocked and immunostained with anti-CD3z, anti-Golgin and anti-LAMP-1 antibodies. Representative images of three independent experiments are shown. The extent of colocalization of CD3z with LAMP-1 (d) or with Golgin (e) was analyzed by ImageJ software. Over 50 control and LAPTM5-expressing cells at each time point were compared and mean±s.d. of Pearson’s coefficient is presented. *Po0.05 (unpaired t-test). (f) LAPTM5-mediated CD3z degradation was partially inhibited by Brefeldin A, which inhibits the export of newly synthesized proteins from ER to the Golgi. MA5.8 cells expressing CD3z with or without LAPTM5 expression were left untreated or treated with Brefeldin A for 6 h and analyzed for the cellular CD3z levels by intracellular staining. Representative FACS profiles are shown and mean±s.d. of three independent experiments is indicated.
observation that LAPTM5 targets intracellular CD3z but not surface CD3z associated with the mature TCR complex. LAPTM5 and SLAP target CD3z through distinct genetic pathways Earlier studies have revealed that SLAP, an adapter protein specifically expressed in thymic CD4 and CD8 DP cells, is involved in the lysosomal translocation and degradation of CD3z internalized from the cell surface together with other TCR subunits. Consequently, DP cells in SLAP-deficient mice express higher levels of cell surface TCR than do WT DP cells. Our data presented thus far suggest that LAPTM5 promotes the lysosomal translocation of intracellular but not cell surface CD3z, suggesting that LAPTM5 and SLAP function in distinct pathways to mediate CD3z degradation. Consistent with this notion, we found that TCR levels were further elevated on DP cells of SLAP/LAPTM5 double KO (DKO) mice as compared with the SLAP or LAPTM5 singly deficient DP cells (Figures 4a and b). These mice also expressed a transgenic TCR (DO11.10) recognizing the OVA peptide presented by major histocompatibility complex class II Immunology and Cell Biology
molecule, and their DP thymocytes were positively selected to differentiate into CD4 single-positive cells on the H-2d selecting background.29 We found that positive selection of the OVA-specific CD4 þ T cells was further enhanced in DKO mice (Figure 4c). It is interesting to note that TCR levels were only marginally increased in LAPTM5 KO cells compared with WT cells (Figure 4b), whereas TCR levels were more clearly elevated in DKO cells compared with SLAP KO cells (Figure 4b). These observations strongly suggest that LAPTM5 and SLAP function in distinct genetic pathways to mediate CD3z degradation and negatively regulate TCR expression.
DISCUSSION Tight regulation of cell surface TCR expression is critical for normal T-cell development and optimal activation. In the present study we show that the lysosomal protein LAPTM5 targets intracellular CD3z in the Golgi and promotes its lysosomal translocation for degradation while having little affect on the cell surface CD3z associated with the
LAPTM5 targets intracellular CD3z Y Kawai et al 531
merge
TG40 CD3ζ
Golgin
merge
CD3ζ-GRIP WT CD3ζ
MA5.8 CD3ζ
CD3ζ-GRIP WT CD3ζ
Golgin
MA5.8 39±1
TG40 68±8
172±24
492±57
46±5
177±30
Count
448±45
Count
170±13
CD3ζ
CD3ζ-GRIP
CD3ζ
CD3ζ-GRIP WT
merge
182±8 611±41
Ctrl
Count
Golgin CD3ζ-GRIP LAMP-1
Ctrl LAPTM5
Ctrl LAPTM5
LAPTM5
YF 510±45
Count
103±16
CD3ζ
WT LAPTM5 KO SLAP KO DKO
Cell-surface TCR
70
250 200 150 100 50 0
60 % of CD4 SP
Count
Thymic DP cells
Cell-surface TCR (MFI)
Figure 3 A Golgi-localizing CD3z–GRIP fusion protein can be transported to the lysosome and degraded by LAPTM5. (a, b) Subcellular localization of WT CD3z or CD3z–GRIP chimera in MA5.8 (a) or TG40 cells (b). MA5.8 or TG40 cells were transduced with retrovirus expressing pMX-CD3z-IRES-rCD2 or pMX-CD3z-GRIP-IRES-rCD2. The rCD2 þ cells were sorted and stained with anti-CD3z and Golgin. Arrowheads indicate colocalization of CD3z–GRIP with Golgin. Data are representative of three independent experiments. (c, d) LAPTM5-mediated degradation of WT CD3z and CD3z–GRIP in MA5.8 (c) and TG40 (d) cells. MA5.8 (c) or TG40 cells (d) expressing WT CD3z or CD3z–GRIP were transduced with retrovirus expressing pMX-LAPTM5-IRES-hCD8 or pMX-IRES-hCD8 as a control and analyzed for cellular CD3z expression by intracellular staining with an anti-CD3z antibody. Data are representative of four independent experiments and mean±s.d. is shown. (e) Subcellular localization of CD3z–GRIP in control TG40 or TG40 cells expressing LAPTM5. Representative images are shown. Arrowheads indicate colocalization of CD3z–GRIP and LAMP-1. Scale bars, 3 mm. (f) The CD3z YF mutant is equally sensitive to LAPTM5-mediated degradation. MA5.8 cells expressing pMX-CD3z-IRES-rCD2 or pMX-CD3zYF-IRES-rCD2 were transduced with retrovirus expressing pMX-LAPTM5-IRES-hCD8 or pMX-IRES-hCD8 as a control and analyzed for intracellular CD3z levels after gating on hCD8 þ cells. Representative FACS profiles are shown and mean±s.d. of three independent experiments is indicated.
50 40 30 20 10 0
Figure 4 LAPTM5 and SLAP mediate TCR degradation through distinct genetic pathways. (a) Cell surface TCR levels on thymic DP cells from WT, LAPTM5 KO, SLAP KO, and LAPTM5 and SLAP DKO DO11.10 TCR transgenic mice (6-8-week-old age-matched). Data are representative of three independent experiments. (b) The results of three mice of each genotype are depicted. (c) Enhanced positive selection of DP thymocytes in LAPTM5 and SLAP DKO mice. Percentages of CD4 þ CD8 thymocytes of three mice of each genotype are depicted. A horizontal bar indicates the mean. *Po0.05; **Po0.01; ***Po0.001 (one-way analysis of variance þ post hoc test). MFI, mean fluorescence intensity.
TCR complex. Consistent with this finding, we show that the tyrosine phosphorylation of CD3z ITAMs is not required for LAPTM5mediated CD3z degradation. These results suggest that CD3z can
be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3z dependent on TCR signaling, and LAPTM5, which targets intracellular CD3z independent of TCR signaling. Immunology and Cell Biology
LAPTM5 targets intracellular CD3z Y Kawai et al 532
Our findings provide new insights into our understanding of the precise regulation of TCR expression and T-cell activation. We initially found that LAPTM5 promoted the lysosomal translocation of intracellular CD3z but not of cell surface CD3z. Kinetic analysis further revealed that the Golgi appears to be the site where LAPTM5 targets CD3z. It is interesting to note that LAPTM5 itself has been shown to localize in the Golgi before being sorted to the lysosome where it normally resides.30,31 These observations raise the possibility that LAPTM5 promotes the lysosomal translocation of CD3z during its own trafficking from the Golgi to lysosomes. Recently, LAPTM5 has been suggested to transport the mH chain in the pre-BCR complex from the ER to lysosomes.32 It remains to be determined whether the pre-BCR is transported to the Golgi before being sorted to lysosomes. Proteins transported from the ER enter the cis-Golgi network and then exit at the trans-Golgi network, which is a particularly important sorting station among Golgi stacks and directs newly synthesized proteins to different subcellular destinations.33 We speculate that the trans-Golgi network is the discrete site where LAPTM5 functions because the GRIP domain has been suggested to promote targeting of Golgin to the trans-Golgi network34 and the CD3z–GRIP fusion protein could be degraded by LAPTM5. It is possible that LAPTM5 promotes the transport of CD3z into lysosomes by altering its sorting process in the trans-Golgi network. The ITAM is defined by the sequence Yxx(L/I)x6-8Yxx(L/I), and the Y-residues are particularly emblematic as they are phosphorylated and form docking sites for signaling molecules on immunoreceptor triggering. However, we demonstrate that phosphorylation of tyrosines in the ITAMs is dispensable for degradation by LAPTM5 as the WT and CD3z YF mutant are comparably degraded by LAPTM5. These observations suggest that LAPTM5 functions independently of TCR signaling and are well consistent with the result that LAPTM5 preferentially targets intracellular CD3z in the Golgi. ITAM tyrosines are also known to function as an endocytic signal and to promote the internalization and trafficking of CD3z into the lysosome, possibly regardless of phosphorylation status.17 The finding that tyrosine residues of CD3z are not required for LAPTM5-mediated degradation further supports our conclusion that LAPTM5 targets intracellular but not cell surface CD3z associated with the TCR complex. We have previously shown that LAPTM5 deficiency did not affect TCR expression in naive T cells but caused elevated TCR levels after CD3 stimulation. These earlier observations suggested that LAPTM5 function was TCR dependent in primary T cells, a conclusion different from the current study. In our earlier study, primary spleen T cells stimulated with anti-CD3 antibody downmodulated surface TCR, which reached the lowest level at 12 h after stimulation (26). The TCR levels then recovered after 24 h, which correlated with the increased CD3z protein levels (26, 35). Notably, it was during this recovery period that LAPTM5 deficiency led to elevated surface TCR levels, consistent with the current data showing that LAPTM5 targets newly synthesized CD3z. The fact that LAPTM5 deficiency did not affect TCR levels in unstimulated T cells could be due to low levels of TCR metabolism (internalization, recycling and new synthesis) in these cells. Therefore, we think that LAPTM5 preferentially targets newly synthesized CD3z and its function in primary T cells becomes more evident after TCR stimulation, which causes dynamic TCR metabolism and resynthesis of a new TCR complex. Our results, however, do not exclude the possibility that LAPTM5 may also target the internalized TCR complex under certain circumstances in primary cells. Earlier studies have revealed that the adapter protein SLAP and the E3 ubiquitin ligase c-Cbl together regulate TCR levels by targeting the Immunology and Cell Biology
CD3z in the internalized TCR complex for ubiquitination and subsequent lysosomal degradation. The present study shows that LAPTM5 does not affect the fate of the internalized TCR complex but instead targets intracellular CD3z and promotes its lysosomal translocation for degradation. In other words, SLAP/c-Cbl regulates the fate of the fully assembled surface TCR, whereas LAPTM5 regulates the amount of the newly synthesized TCR. Therefore, LAPTM5 and SLAP/c-Cbl mediate CD3z degradation through distinct pathways. This conclusion is further supported by genetic data showing that LAPTM5 and SLAP DKO thymocytes express higher levels of cellular CD3z and surface TCR compared with singly deficient thymocytes. The DKO mice also show enhanced positive selection of the DP thymocytes, suggesting that the elevated TCR levels indeed result in increased TCR signaling required for efficient positive selection and differentiation into the more mature singlepositive thymocytes. It has been shown that CD3z protein levels decrease first and then increase at late time points after TCR signaling.35 The time course of CD3z expression correlates with the cell surface TCR levels. Notably, LAPTM5 protein levels also increase at late time points after T-cell activation, presumably to prevent overshooting of normal TCR expression levels, which would lead to excessive and/or prolonged T-cell activation. In fact, our earlier studies have revealed that TCR levels on LAPTM5 KO spleen T cells become much higher than those on WT spleen T cells only at late time points (24 h and 48 h) after TCR crosslinking.26 Collectively, the LAPTM5 pathway functions at a relatively late phase when T cells start to recover from their downmodulated TCR expression by assembly of newly synthesized TCR subunits, in contrast to the SLAP/c-Cbl pathway that functions during an early phase of TCR signaling after TCR internalization. Therefore, T cells likely possess at least two layers of regulatory mechanisms that function at different time points and in distinct cellular locations to strictly control surface TCR expression for normal T-cell development and activation. The present study has revealed a new pathway that promotes CD3z degradation. It has been reported that abnormal TCR lacking some of the TCR subunits could reach and reside on the cell surface to some extent, partially overcoming the developmental block at the positive selection stage, as was observed in CD3g-, d- and z-deficient mice.36–38 In addition, earlier studies demonstrated that high-level expression of human CD3d rescued TCR expression in CD3g- and CD3d-deficient mice,39 raising the possibility that some TCR subunits, if produced in excess, could be substituted for deficient subunits in the formation of an abnormal TCR complex. Cell surface expression of the partially assembled receptor in the absence of one of the receptor components may result in the generation of autoreactive T cells.40,41 In this regard, it will be interesting to determine whether LAPTM5 suppresses abnormal TCR expression by degrading excessive intracellular TCR subunits during TCR assembly. METHODS Mice LAPTM5 KO, SLAP KO and DO11.10 TCR transgenic mice have been described previously.24,26 All the mice were maintained under specific pathogen-free conditions and all experiments were approved by the Animal Facility Committee of RIKEN Yokohama Institute (Permission number 20-025).
Antibodies and other reagents The following antibodies were used: anti-LAPTM5,26 anti-LAMP-1 (1D4B, BD Biosciences, San Jose, CA, USA) and anti-Golgin-97 (CDF4, Molecular Probes) for immunostaining; anti-TCRb (H57-597, Biolegend, San Diego, CA,
LAPTM5 targets intracellular CD3z Y Kawai et al 533 USA), anti-CD3e (145-2C11, Biolegend), anti-CD4 (GK1.5, Biolegend), antiCD8 (Ly-2, Biolegend), anti-rat CD2 (OX-34, BD Biosciences) and antihuman CD8 (RPA-T8, eBioscience, San Diego, CA, USA) for flow cytometry; anti-ACTIN (rabbit polyclonal antibodies, Sigma, St Louis, MO, USA) and anti-CD3e (M-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for western blotting; anti-CD3z mAb (H146) used in all applications in this study was a kind gift from Dr T. Saito (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan).
Cells and culture Both MA5.8 and TG40 are variants of the T-cell hybridoma 2B4.11 specific for I-Ek-restricted cytochrome c peptide.10,28 These cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, 50 mM 2-mercaptoethanol and antibiotics. The PHOENIX packaging cell line was maintained in Dulbecco’s modified Eagle medium medium supplemented with 10% fetal calf serum and antibiotics.
Construction and retroviral transduction The CD3z–GRIP chimera consists of full-length CD3z C-terminally fused to 100 amino acids of Golgin-97 (659–759). YF mutant of CD3z, in which all the six Tyr in three ITAMs are mutated to Phe, was generated by site-directed mutagenesis using the PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc., Shiga, Japan). DNA fragments encoding these proteins including WT CD3z and LAPTM5 were inserted into the EcoRI/XhoI site of pMX-IRES-rCD2, pMX-IRES-hCD8 or pMX-IRES-GFP retroviral vector. The retroviral constructs were transfected into the PHOENIX packaging cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and the viral supernatant was prepared as described previously.26 Virus transduction was carried out in the presence of 10 mg ml 1 DOTAP (Roche, Basel, Switzerland). In experiments analyzing the effect of LAPTM5 on CD3z/CD3e localization and degradation, MA5.8 or TG40 cells were transduced with retrovirus expressing either LAPTM5 or the transduction marker (rCD2, hCD8 or GFP) alone as a control and cells with similar levels of the transduction marker were sorted. Appropriate combinations of retroviruses were used in transduction of MA5.8 or TG40 cells as indicated in the figure legend.
Immunostaining Cells were fixed with 6% formalin/phosphate-buffered saline (PBS) for 15 min at room temperature, permeabilized with 0.1% Triton-X-100/PBS for 5 min, washed, blocked with 1% bovine serum albumin/PBS and incubated with primary antibodies at room temperature for 1 h followed by the Alexa Fluor 488-, 555- or 647-conjugated secondary antibodies for 30 min. The cells were examined under a confocal microscope (SP2AOBS, Leica Microsysytems, Wetzlar, Germany), and photographs were recorded with Leica confocal software.
Flow cytometry Multicolor flow cytometric analysis was performed with FACSCalibur (BD Biosciences). Analysis of intracellular molecules was performed as follows: cells were fixed with 6% formalin/PBS for 10 min at 37 1C, permeabilized with 0.2% Tween 20/PBS for 20 min, washed, blocked with 1% bovine serum albumin/ PBS and incubated with staining antibodies at room temperature for 20 min. The cellular CD3z was stained with H146 antibody followed by PE-conjugated anti-hamster IgG.
Immunoprecipitation and immunoblotting Cells were lysed at 4 1C for 10 min in lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA and complete protease inhibitor cocktail tablets (Roche) containing 1% NP-40) and were centrifuged at 20 000 g for 5 min, following which the supernatant was recovered. The lysates were incubated with indicated biotinylated antibodies at 4 1C for 2 h, followed by incubation with streptavidin sepharose (GE Healthcare, Little Chalfont, UK) at 4 1C for 30 min, and the immunoprecipitates were washed four times with lysis buffer, resuspended with sample buffer and boiled for 5 min. The samples were resolved by PAGE, transferred to immobilon-P membranes
(Millipore, Billerica, MA, USA), blocked with blocking one (Nacalai, Kyoto, Japan), incubated with the indicated primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, and then developed with Immobilon Western Chemiluminescent horseradish peroxidase substrate (Millipore).
Statistical analysis Statistical significance was assessed with the unpaired Student t-test for Figure 2 and analysis of variance þ post hoc test for Figure 4. *Po0.05; **Po0.01; ***Po0.001.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We are grateful to Drs T. Saito and T. Yokosuka for providing the MA5.8 and TG40 cells and the anti-CD3z mAb. We also thank Drs A. Weiss, S. Fagarasan, H. Kawamoto, K. Masuda, K. Kakugawa and K. Kometani for helpful suggestions, the Animal Facility for maintaining the mice and the FACS Facility for cell sorting.
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