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Previous Article | Next Article 
Molecular and Cellular Biology, December 2007, p. 8622-8636, Vol. 27, No. 24
0270-7306/07/$08.00+0 doi:10.1128/MCB.00467-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
,
Amrita Goyal,
Matthew S. Isakowitz, and
Lawrence E. Samelson*
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Received 19 March 2007/ Returned for modification 8 May 2007/ Accepted 27 September 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 (PLC-1), the Rho family GTPase exchange factor Vav, and the ubiquitin ligase Cbl (48). Thus, TCR engagement induces the formation of LAT-based signaling complexes that initiate intracellular signals required for T-cell activation. Modern imaging techniques have given us insights into the dynamics of signaling proteins within a single cell. Studies from our lab using immobilized stimulatory antibodies binding the TCR showed that the TCR-rich microclusters form within seconds of activation and rapidly recruit several other signaling proteins (5, 8, 10). Similar microclusters have been observed in T cells activated by antigen-presenting lipid bilayers and antigen-presenting cells (11, 49). Importantly, microclusters are the principal sites of TCR-induced tyrosine phosphorylation and form coincident with the initial calcium response in a T cell, indicating that signaling is initiated at these clusters (10, 11).
To ensure an appropriate immune response to antigenic challenge, without generating an autoimmune response, it is crucial that T-cell activation be tightly regulated. TCR engagement activates several mechanisms that have been described to attenuate TCR-mediated signaling (46), including ligand-induced internalization and degradation of activated signaling molecules (25). For example, c-Cbl-mediated ubiquitin conjugation to the TCR
chain has been correlated with TCR internalization into endosomal compartments and the subsequent degradation of the receptor in activated T cells (17). In addition, Cbl proteins downregulate PTKs such as Lck, Fyn, and ZAP-70 (3, 34, 42), as well as non-PTK molecules such as the p85 subunit of phosphatidylinositol 3-kinase and the guanine nucleotide exchange factor Vav (16, 30). Furthermore, in anergic T cells, Cbl-b-mediated ubiquitylation is thought to contribute to early destabilization of the immune synapse and therefore lead to decreased signaling (21, 26). Thus, Cbl-mediated protein ubiquitylation in T cells has emerged as an important mechanism to attenuate the T-cell response.
While most studies on internalization as a means of signal downregulation in T cells have focused on the fate of the TCR, results from studies tracking individual components of TCR-induced microclusters in real time suggest that the fates of the TCR and signaling proteins diverge during T-cell activation. In systems using either stimulatory antibodies or lipid bilayers to model T-cell activation, whereas microclusters contain both the TCR and signaling molecules initially, signaling molecules dissociate from the receptor soon thereafter (10, 11, 15, 49). Consistent with these observations, immunoelectron microscopy analysis of mast cells has shown that upon activation, mast cell membranes are organized into primary signaling domains, which are formed around the Fc
receptor I and associate with clathrin-coated pits, and secondary signaling domains, which are organized around LAT and do not include the receptor or coated pits (47). Thus, at least in some cell types, receptor and LAT-nucleated signaling domains are discrete, and the internalization of proteins in the two domains appears to be regulated by distinct mechanisms. Although numerous studies have examined receptor internalization, not much is known about the internalization and fate of signaling molecules, such as LAT and SLP-76. A recent study from our group demonstrated that the cytosolic adapter molecule SLP-76 is endocytosed in vesicles that also contain a percentage of cellular LAT. SLP-76 endocytosis occurs through a lipid raft-dependent pathway that requires the association of the endocytic machinery with ubiquitylated proteins (6). Of interest, a recent study reported that LAT is a target of ubiquitylation (9), raising the possibility that LAT may be the ubiquitylated component that drives the internalization of the LAT-SLP-76 complex. A previous study from this group demonstrated that LAT is observed in endosomal compartments (7). However, the correlation between LAT ubiquitylation and LAT internalization remains unclear.
In the present study, we demonstrate that upon TCR activation, LAT-containing signaling clusters are internalized into various distinct intracellular compartments prior to dissipating rapidly. To investigate the molecular mechanism that regulates both the internalization and dissipation of signaling clusters, we examined the role of the ubiquitin ligase c-Cbl in signaling complex internalization. We expressed versions of c-Cbl that were defective in the RING finger domain, which mediates ubiquitin ligase activity, and assessed their effect on the trafficking of TCR-induced protein complexes. The expression of ligase-defective Cbl mutants resulted in severely decreased internalization of LAT and SLP-76 clusters, decreased ubiquitylation of LAT, and an increase in basal LAT levels, as well as elevated basal and TCR-induced phosphorylated LAT (pLAT) levels. The inhibition of LAT internalization was also observed in T cells from mice lacking c-Cbl. Our data are consistent with a model in which TCR-mediated activation first leads to the rapid formation of signaling complexes, after which c-Cbl activity is involved in the internalization and possible downregulation of a subset of activated signaling molecules.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, anti-CD28, and anti-CD4 and human anti-CD3 (UCHT or HIT3a) were all purchased from BD Pharmingen. OKT3 against the human CD3 chain was used to trigger T-cell activation. The following antibodies were used for immunofluorescence: anti-c-Cbl (BD Transduction Labs), rabbit anti-LAT (described in reference 51), antibodies to LAT phosphorylated at tyrosine 191 and to ZAP-70 phosphorylated at tyrosine 315 and 319 (Biosource International), antiphosphotyrosine (anti-pY) 4G10 (Upstate), anti-SLP-76 (Antibody Solutions), and anti-TCR 6B10 (Santa Cruz Biotechnologies). The following antibodies were used for Western blotting and immunoprecipitation: antihemagglutinin (anti-HA)-horseradish peroxidase (Roche), mouse anti-LAT (Upstate), anti-c-Cbl (BD Transduction Labs), anti-glyceraldehyde-3-phosphate dexhydroxygenase (anti-GAPDH; Biodesign International), and antibody to LAT phosphorylated at tyrosine 191 (Biosource International). Isotype-specific secondary antibodies and Alexa 594-labeled transferrin and cholera toxin subunit B (CTX-B) were obtained from Molecular Probes. Expression vectors and plasmids. The expression vectors pEYFP-N1 and pECFP-N1 were obtained from Clontech. The plasmid pCDNA3.1+ Hygro was obtained from Invitrogen. The SLP-yellow fluorescent protein (YFP) and LAT-YFP constructs have been described previously (10). The c-Cbl cDNAs from the pSX HA Cbl and pSX HA 70Z-Cbl constructs described previously (43) were cloned into the pEYFPN1 expression vector to generate the wild-type (WT) Cbl-YFP and the 70Z/3 Cbl-YFP constructs, respectively. For cyan fluorescent protein (CFP)-tagged constructs, the c-Cbl coding sequences were cloned into the pCDNA3.1+ Hygro vector. Point mutations and deletions were introduced into Cbl constructs by using a QuikChange II XL site-directed mutagenesis kit (Stratagene). All constructs were verified using DNA sequencing. The following constructs were expressed in COS-7 cells: the HA-tagged ubiquitin plasmid was provided by Dirk Bohmann, and the myc-tagged LAT construct (51) and the pSX FLAG-Cbl construct (43) have been described previously.
Cell culture and transfection of Jurkat T cells. Jurkat E6.1 cells, LAT-deficient JCam2.5 cells, and reconstituted variants of JCam2.5 cells have been described previously (53); ZAP-70-deficient P116 cells and P116 reconstituted variants have also been described previously (44). All Jurkat cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics. For protein expression, Jurkat cells were transfected with the 5- to 10-µg plasmid DNA by using the electroporation system, solution T, and program H-10 from Amaxa Biosystems. Transiently transfected cells were used 24 to 48 h posttransfection. For the generation of stable cell clones, transiently transfected cells were subjected to drug selection, followed by cell sorting. Single-cell clones were then obtained by plating sorted cells at limiting dilutions.
Transfection of murine T cells. Mice used in this study were on the C57BL/6 background. c-Cbl–/– mice have been described previously (32). CD4+ T cells from the lymph nodes of wild-type (WT) C57BL/6, c-Cbl–/–, or c-Cbl–/+ mice were purified by magnetic bead separation as previously described (37). Cells were transfected with LAT-YFP in the case of c-Cbl–/– and c-Cbl–/+ T cells and with WT Cbl-YFP or 70Z/3 Cbl-YFP in the case of the C57BL/6 T cells by using the Amaxa electroporator, the Amaxa kit for primary murine T cells, and program X-01. Cells were stimulated on coverslips 24 h posttransfection and processed as described in "Confocal microscopy" below.
Confocal microscopy.
Spreading assays were performed as described previously (10). Briefly, chambered coverslips (LabTek) were coated overnight at 4°C with the stimulatory antibody human anti-CD3 (HIT3a or UCHT at 10 µg/ml) or mouse anti-CD3, anti-CD4, and anti-CD28 (10 µg/ml each). Cells were plated onto coated coverslips containing imaging buffer (RPMI 1640 without phenol red, 10% fetal calf serum, 20 mM HEPES). Cells were fixed at different time points with 2.4% paraformaldehyde. Immunostaining was performed as described previously (5). Fluorescent images of fixed samples were acquired on a 510 laser-scanning confocal microscope system by using a 63x plan apochromat objective (Carl Zeiss). The movement of fluorescent proteins in live cells was observed with a Zeiss Axiovert 200 microscope equipped with a PerkinElmer Ultraview spinning-disk confocal system (PerkinElmer). Images were captured with an Orca-ERII charge-coupled device camera (Hamamatsu). A hot air blower and an objective warmer were used to maintain live samples at 37°C.
Image processing. IPLab 3.6 (Scanalytics Inc.) was used for most image processing. Movies were prepared from z-stacks by making a maximum-intensity projection for a given time point and then making a sequence of all the projections. Kymographs were made from regions of interest drawn around moving clusters of interest, and the movement of clusters was analyzed using IPLab 3.6. Graphs were prepared with Microsoft Excel. Adobe PhotoShop and Adobe Illustrator (Adobe Systems Inc.) were used to prepare composite figures.
siRNA-mediated depletion of LAT levels. The small interfering RNA (siRNA) corresponding to human LAT and the control nontargeting siRNA pool were purchased from Dharmacon Inc. The SMARTpool duplexes for human LAT were designed to target the following cDNA sequences: SMARTpool duplex 1, GCACAUCCUCAGAUAGUUUUU; duplex 2, CAAACGGCCUCAACGGUUUU; duplex 3, GGACGACUAUCACAACCCAUU; and duplex 4, CCAACAGUGUGGCGAGCUAUU. Briefly, Jurkat cells were transfected with control siRNA or siRNA for LAT (100 µm/5 x 106 cells) by using an Amaxa electroporator, Amaxa solution T, and program H-10. Forty-eight hours after electroporation, cells were lysed in ice-cold lysis buffer containing 1% Brij, 1% octyl-ß-D-glucoside, 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, and a complete protease inhibitor tablet (Roche). Lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membrane, and immunoblotted for LAT and GAPDH. Alternatively, cells were plated onto stimulatory coverslips and processed as described under "Confocal microscopy" above.
COS-7 cell transfection, immunoprecipitation, and immunoblotting. COS-7 cells were transfected using Lipofectamine Plus reagent as recommended by the manufacturer (Sigma). Briefly, 60-mm dishes with 70% confluent cell cultures were used for transfection. Cells were cotransfected with plasmids indicated in the figures, and 24 h posttransfection, cells were lysed in ice-cold NP-40 lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 50 mM NaF, 1% NP-40) and cellular lysates were subjected to immunoprecipitation using mouse anti-LAT monoclonal antibody (Upstate). Protein A/G Plus-agarose beads (Santa Cruz Biotechnology) were used for immunoprecipitation. Protein samples were resolved by SDS-PAGE, transferred onto PVDF membrane, and subjected to immunoblotting with primary antibodies followed by enhanced chemiluminescence with a system from Upstate. The denaturation of immunoprecipitated complexes and the reprecipitation of LAT were performed as described previously (27). Briefly, the first immunoprecipitation was performed as described above. The precipitated complex was resuspended and boiled for 5 min in 40 µl of denaturation buffer (20 mM Tris-HCl [pH 7.4], 50 mM NaCl, 5 mM dithiothreitol, 1% SDS, and 1 mM sodium orthovanadate). The denatured sample was diluted to 800 µl with lysis buffer, and the second immunoprecipitation was performed, again as described above.
Cell stimulation and immunoblotting of Jurkat cells. Jurkat E6.1 cells stably expressing LAT-YFP were transiently transfected with CFP, WT Cbl-CFP, or 70Z/3 Cbl-CFP. Twenty-four hours after transfection, cells containing both CFP and YFP were sorted. Sorted cells were washed once with RPMI 1640 without supplements and resuspended to a concentration of 5 x 107 cells/ml. The cells were then treated with anti-CD3 (OKT3 ascites fluid; 1/200) for various time periods and lysed using a twofold excess of hot 2x sample buffer (20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 2 mM Na3VO4, 20 mM dithiothreitol, 2% SDS, and 20% glycerol). The samples were then heated to 95°C for 5 min and sonicated to reduce the viscosity of the solution. To analyze the phosphorylation status of LAT, 2.4 x 105 cell equivalents were separated by SDS-PAGE. Separated proteins were then transferred onto PVDF membrane and immunoblotted for pLAT, total LAT, GAPDH, and HA.
Flow cytometric assays. Cells stably expressing LAT-YFP were transfected with various WT and 70Z/3 Cbl-CFP constructs. Twenty-four hours after transfection, cells were analyzed using a Becton Dickinson FACSVantage SE flow cytometer (Becton Dickinson Inc.). The data were analyzed with FloJo software. Mean LAT-YFP levels (± standard errors of the mean [SEM]) were measured in the CFP+ YFP+ quadrant and were normalized to levels of the CFP vector control.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Persistence and movement of LAT clusters can be modulated by c-Cbl RING activity. We next sought the molecular mechanism that could mediate both the internalization of LAT and the loss of visible LAT-YFP clusters upon TCR activation. Since the ubiquitin ligase c-Cbl is involved in endocytic trafficking of the TCR and the downregulation of several proteins involved in T-cell signaling (13), we decided to examine whether c-Cbl played a role in LAT cluster internalization and dissipation. Fixed-cell analysis revealed that c-Cbl is recruited to the signaling clusters that contain LAT and SLP-76 upon the activation of the TCR (data not shown) (10). To examine the effect of c-Cbl expression on LAT dynamics in living cells, we transiently transfected E6.1 LAT-YFP cells with either WT Cbl-CFP or 70Z/3 Cbl-CFP, an oncogenic, dominant negative version of c-Cbl with a 17-amino-acid internal deletion in the RING finger domain that abolishes ubiquitin ligase activity (4). Figure 2 shows the movement of LAT-YFP in a cell that has been transfected with CFP vector only (Fig. 2A), a cell coexpressing WT Cbl-CFP (Fig. 2B), or a cell coexpressing 70Z/3 Cbl-CFP (Fig. 2C; see Videos S4 to S6 in the supplemental material). As expected, CFP appeared to be cytosolic and did not get recruited into clusters (Fig. 2A, bottom panels). In comparison, WT Cbl-CFP clustered rapidly at the signaling complexes, and Cbl clusters dissipated soon thereafter (Fig. 2B, bottom panels). In the presence of CFP vector or WT Cbl-CFP, LAT-YFP was rapidly recruited into clusters, after which clusters containing LAT dissipated within 2 min (Fig. 2A and B, top panels). Of note, the central LAT structure that persisted in the cell expressing WT Cbl-CFP (Fig. 2B, top panel) was LAT-YFP localized in the Golgi compartment, as evaluated by colocalization with Golgi markers (data not shown). In contrast, 70Z/3 Cbl-CFP clusters remained visible longer and did not dissipate (Fig. 2C, bottom panels). Remarkably, in cells expressing 70Z/3 Cbl, LAT-YFP clusters persisted, colocalized with 70Z/3 Cbl-CFP clusters for extended periods of time, and failed to translocate (Fig. 2C, top panels).
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TCR-induced activation leads to the phosphorylation of LAT, which creates docking sites for the recruitment of several proteins, including SLP-76. Additionally, since a pool of LAT is found in vesicles containing SLP-76, we assessed the effect of Cbl expression on SLP-YFP clusters. We transiently transfected E6.1 Jurkat cells stably expressing SLP-YFP with CFP-tagged constructs. Similar to its effect on LAT-YFP clusters, 70Z/3 Cbl-CFP caused persistence and retarded movement of SLP-YFP clusters, while in the presence of additional WT Cbl, the SLP-YFP clusters faded more rapidly (Fig. 2F and G; also see Videos S10 to S12 in the supplemental material). Since LAT and SLP-YFP are rapidly internalized upon T-cell activation, these data support the conclusion that c-Cbl ubiquitin ligase activity, mediated by the RING domain, is required for the sorting of LAT and SLP-76 into mobile, endocytic structures.
To verify that the effect of Cbl on LAT clusters occurred in nontransformed cells, we transfected primary murine CD4+ lymph node cells from C57BL/6 mice with either WT Cbl-YFP or 70Z/3 Cbl-YFP and dropped the cells onto stimulatory coverslips. We fixed the cells at various times after activation and immunostained them for LAT. Soon after activation, signaling clusters that contained LAT and WT Cbl-YFP or 70Z/3 Cbl-YFP were observed (Fig. 3A). Similar to the phenotype in Jurkat cells, we saw significant differences in the persistence of LAT and Cbl clusters at later time points. In cells that were transfected with WT Cbl-YFP, both Cbl and LAT clusters had largely dissipated by 15 min (Fig. 3B, top panel). In contrast, cells containing 70Z/3 Cbl-YFP displayed persistent LAT and 70Z/3 Cbl clusters (Fig. 3B, bottom panel, and quantified data shown in Fig. 3C), thus extending the observations made with YFP-tagged LAT in Jurkat cells to endogenous LAT in primary cells.
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Requirement of LAT and binding to ZAP-70 for c-Cbl recruitment to activation clusters.
Having determined the domain requirements of c-Cbl for the localization of c-Cbl to TCR-induced clusters, we next investigated which proteins at the clusters are important in c-Cbl recruitment. To evaluate the importance of LAT and LAT-interacting proteins, we examined c-Cbl localization in JCam2.5 cells that lack LAT or JCam2.5 cells reconstituted with LAT mutant forms that lack either the three Grb2 binding sites (LAT3YF) or the PLC- binding site (LATY132F). These cells were dropped onto stimulatory coverslips, fixed soon after activation, and immunostained for pY to mark the activation clusters, pLAT to mark LAT clusters, and c-Cbl to assess c-Cbl recruitment (Fig. 6A). JCam2.5 cells reconstituted with wild-type LAT served as a positive control (Fig. 6A, top panel). Of note, there appears to be no pLAT at the activation clusters in the JCam2.5 and JCam2.5 LAT3YF cells, indicating that, as we reported previously, LAT clusters do not form in these cells (Fig. 6A, panels 2 and 4) (24). To our surprise, c-Cbl was localized at the signaling clusters in all the cell lines examined. Since LAT was not essential for the recruitment of c-Cbl to signaling clusters, we next analyzed which other proteins may be important in c-Cbl localization.
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Next, we considered whether LAT and ZAP-70, together, accounted for c-Cbl recruitment to clusters. To evaluate this possibility, we simultaneously eliminated LAT and ZAP-70 binding by using an siRNA pool to reduce LAT expression in P116 cells reconstituted with ZAP-70 Y292F. In cells transfected with siRNA pools targeting LAT, LAT expression was reduced by 80% (data not shown). A control siRNA pool was used to control for off-target effects. The siRNA-mediated inhibition of LAT expression in P116 cells reconstituted with WT ZAP-70 did not lead to an inhibition of c-Cbl clustering (Fig. 6C). In contrast, LAT depletion in P116 cells reconstituted with ZAP-70 Y292F led to a significant decrease in the number of cells with distinct c-Cbl clusters (Fig. 6D; quantification in panel E). Of note, the absence of pLAT staining in these cells confirmed the effective knockdown of LAT expression. These data indicate that although the binding of LAT and ZAP-70, taken separately, is dispensable for c-Cbl clustering, the loss of both these signaling components abolishes c-Cbl recruitment to activation clusters.
LAT is ubiquitylated, and 70Z/3 Cbl expression suppresses LAT ubiquitylation. Since c-Cbl functions as a ubiquitin ligase, we next investigated whether molecules affected by the expression of c-Cbl RING mutant proteins in our imaging assay were potential substrates of c-Cbl ubiquitin ligase activity. For this purpose, we tested for the ubiquitylation of the signaling molecules LAT and SLP-76 in COS-7 cells. COS-7 cells were transfected with HA-tagged ubiquitin and SLP-76 or LAT, followed by the immunoprecipitation of SLP-76 or LAT and anti-HA Western blotting. Consistent with the findings of a recently published study that presented evidence for LAT ubiquitylation (9), the immunoprecipitation of LAT resulted in the coprecipitation of ubiquitylated bands (Fig. 7). In contrast, no HA-tagged ubiquitin was detected in SLP-76 immunoprecipitates (data not shown). Notably, in the LAT immunoprecipitates, the predominant ubiquitylated band was detected at around 48 kDa (Fig. 7A, top panel). Since immunoprecipitated LAT runs at 40 kDa, this band likely represents monoubiquitylated LAT. The bands above the 48-kDa band may represent multimonoubiquitylated or polyubiquitylated LAT species. To confirm that the ubiquitylated bands detected by LAT immunoprecipitation were in fact modified LAT and not LAT-associated proteins, we immunoprecipitated LAT and denatured the immunoprecipitated proteins, allowed them to be renatured, and then reimmunoprecipitated LAT. As seen in Fig. 7B, the ubiquitylated bands were detected in both the first and second LAT immunoprecipitations, indicating that the bands detected by the HA antibody included ubiquitylated LAT species.
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70Z/3 Cbl regulates basal cellular LAT levels as well as basal and TCR-induced LAT phosphorylation. The microscopic and biochemical data described above demonstrate that c-Cbl activity modulates both LAT internalization and LAT ubiquitylation. To assess whether Cbl might have an effect on LAT expression, we developed a system to evaluate basal LAT levels in cells expressing various Cbl constructs. In this system, a stable cell line expressing LAT-YFP was transfected with Cbl-CFP, 70Z/3 Cbl-CFP, or a construct expressing CFP alone as a control. Following transfection, LAT-YFP levels in transfected (CFP+) cells were assessed by flow cytometry. LAT-YFP levels were not substantially altered in cells transfected with Cbl-CFP compared with those in CFP control-transfected cells. To our surprise, in cells transfected with 70Z/3 Cbl-CFP, LAT-YFP levels were significantly increased (Fig. 8A and B). Quantification of these data revealed that LAT-YFP levels were increased by more than three times in cells expressing 70Z/3 Cbl-CFP compared to levels in cells expressing a CFP vector control (Fig. 8B). Importantly, 70Z/3 Cbl-CFP expression did not cause an increase in YFP levels in control cells stably expressing YFP, indicating that the increase observed in LAT-YFP levels is not due to the global upregulation of expressed plasmids mediated by 70Z/3 Cbl (data not shown).
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We were also interested in monitoring the effects of c-Cbl expression on LAT levels following activation. However, we could not detect any changes in total LAT protein levels upon OKT3-induced TCR triggering, either by Western blotting or by flow cytometry (Fig. 8C and data not shown). One possible explanation is that only a small fraction of LAT is recruited to the activation clusters and subjected to c-Cbl-dependent ubiquitylation and degradation. Therefore, to better understand the fate of activated pools of LAT, we examined levels and kinetics of LAT phosphorylation using a phospho-specific antibody to tyrosine 191 of LAT. Upon TCR stimulation, tyrosine 191 of LAT gets phosphorylated rapidly, reaching maximal stimulation at 60 s (23). To evaluate the effects of c-Cbl expression on pLAT, E6.1 LAT-YFP cells were transfected with CFP, WT Cbl-CFP, or 70Z/3 Cbl-CFP. Following transfection, cells were stimulated and the degree of LAT phosphorylation was determined at various time points. In cells transfected with CFP and WT Cbl-CFP, pLAT levels peaked at 60 s and decreased slightly by 120 s (Fig. 8C and D), though pLAT levels in WT Cbl-CFP-transfected cells were comparatively lower at both time points. In contrast, 70Z/3 Cbl-CFP expression augmented both basal and TCR-induced pLAT levels. Furthermore, pLAT levels remained elevated at 120 s. These results suggest that c-Cbl function is involved in regulating pools of pLAT and that, upon TCR triggering, c-Cbl activity regulates the loss of phosphorylated LAT protein. Importantly, these data suggest that the inhibition of LAT ubiquitylation and LAT cluster endocytosis by 70Z/3 Cbl have consequences for LAT signaling.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Importantly, we have uncovered a role for the ubiquitin ligase c-Cbl in the internalization of LAT-nucleated clusters. The expression of versions of c-Cbl that are defective in the RING domain severely inhibited LAT and SLP-76 movement and caused the persistence of signaling clusters. These data suggest that c-Cbl ubiquitin ligase activity is intimately involved in the sorting of LAT and SLP-76 into mobile endocytic structures. Though the function of c-Cbl in the endocytosis of immune and growth factor receptors has been well documented, the novelty of our demonstration is that this mechanism can regulate activated signaling complexes independently of the TCR, which does not accompany LAT and SLP-76. Additionally, LAT internalization appears to be inhibited in CD4+ cells from c-Cbl–/– mice, confirming that the effects of 70Z/3 Cbl expression reflect c-Cbl function. We note that the extent of inhibition of LAT internalization by 70Z/3 Cbl expression in CD4+ lymph node cells is greater than that caused by the lack of c-Cbl function in CD4+ cells from c-Cbl–/– mice. It is possible that in addition to inhibiting c-Cbl function, 70Z/3 Cbl expression inhibits the function of other Cbl family members or even other proteins. Alternatively, the biology of the peripheral CD4+ cells from the c-Cbl–/– mice may differ from that in wild-type CD4+ T cells used in the 70Z/3 Cbl experiments, due to altered signaling in the c-Cbl–/– thymocytes undergoing selection, thus effecting the number of mature T cells that exhibit persistent LAT clusters (32).
To give us insight into the mechanisms by which c-Cbl regulates signaling cluster dynamics, we examined the requirements for domains in c-Cbl, as well as the proteins at the cluster required for c-Cbl recruitment to TCR-induced clusters. The importance of c-Cbl proline-rich and TKB domains in c-Cbl recruitment and stabilization at signaling clusters is compatible with a role for LAT and ZAP-70. LAT interaction with the c-Cbl proline-rich domain can be mediated by Grb-2, PLC-, or NCK (36), and ZAP-70 interacts with the TKB domain of c-Cbl (29). Interestingly, though neither the binding of LAT nor that of ZAP-70 was required individually for c-Cbl recruitment, the simultaneous depletion of both molecules abolished c-Cbl recruitment to clusters. These data indicate a redundancy in the system and show that the presence of either ZAP-70 or LAT is sufficient to stabilize c-Cbl recruitment to the same TCR-induced clusters, without the participation of the other. Alternatively, it is possible that ZAP-70 and LAT are at distinct signaling clusters and that the elimination of either signaling component allows c-Cbl to be recruited into signaling clusters containing the other protein. Consistent with this scenario, in mast cell membranes, receptor-containing signaling domains and LAT-containing domains are discrete (47).
The requirement for the c-Cbl RING domain in the internalization of signaling clusters prompted us to look for the ubiquitylation of molecules that were inhibited by RING finger mutant forms of c-Cbl. Consistent with a recent report (9), we observed the modification of LAT by ubiquitylation. Furthermore, we have demonstrated that LAT ubiquitylation can be modulated by c-Cbl, with WT Cbl expression causing an increase and 70Z/3 Cbl expression causing a decrease in ubiquitylated LAT species. In recent years, it has become clear that, in addition to its well-known role in proteasome-dependent protein degradation, ubiquitin is involved in protein trafficking and membrane protein internalization (1, 22). Thus, c-Cbl-mediated LAT ubiquitylation may serve as a regulated signal for the internalization of LAT and perhaps LAT-nucleated signaling complexes.
However, c-Cbl may regulate LAT endocytosis by other mechanisms. One possibility is that effects seen with the Cbl mutants on endocytosis result from the defective ubiquitylation of proteins other than LAT. These proteins may potentially be other signaling molecules in the LAT-nucleated signaling complex. Alternatively, endocytic adapter proteins may be the targets of ubiquitylation. In fact, it has been proposed that the ubiquitylation of a component of the endocytic apparatus is required for the endocytosis of 
factor receptor in yeast (14) and growth hormone receptor in mammalian cells (18). Finally, it is possible that the RING domain of Cbl is required to recruit proteins essential for the endocytosis of microclusters in a ubiquitin-independent manner.
The loss of LAT clusters with time and the regulation of cellular LAT levels by c-Cbl suggests that the dissipation of LAT represents degradation. Interestingly, stimulation of the TCR is not required for the observed increase in LAT levels. A significant amount of data support the idea that some basal level of signaling occurs continuously in most signal-transducing systems and is the result of an equilibrium between positive and negative regulators of a signaling pathway (35). Our data suggest that the expression of 70Z/3 Cbl perturbs the function of the negative regulatory protein c-Cbl, thus uncovering a role for the c-Cbl RING domain in the regulation of homeostatic basal LAT levels in resting cells.
Although LAT-YFP clusters dissipate rapidly upon TCR stimulation, we did not detect a decrease in LAT protein levels following T-cell activation in the defined time course over which we observed the loss of LAT clusters. One possible explanation is that only a small fraction of cellular LAT is recruited into signaling complexes, internalized, and degraded. Therefore, we decided to monitor the effects of c-Cbl expression on the activated pool of pLAT. Interestingly, we saw an increase in basal and activation-induced levels of pLAT upon 70Z/3 Cbl expression. Similarly, thymocytes from both c-Cbl–/– mice and mice with a loss-of-function mutation in the c-Cbl RING domain exhibited increased and sustained phosphorylation of LAT and SLP-76, probably due to deregulated ZAP-70 activity (39, 40). In our studies, we cannot rule out the possibility that the effects on LAT internalization and levels by c-Cbl are mediated through an effect on ZAP-70. However, the observations that LAT is ubiquitylated and that c-Cbl modulates LAT ubiquitylation are suggestive of direct regulation of LAT by c-Cbl.
Current spatial models of TCR-induced microcluster signaling and downregulation propose that the central supramolecular activation cluster (cSMAC) is a site for TCR downregulation and signal attenuation (28, 45). In our model system, TCR microclusters remain immobilized by the stimulatory antibody on the coverslip, and hence, we do not observe cSMAC formation. However, in addition to the degradation of the TCR at the cSMAC, the regulation of signaling probably also occurs at the level of the microclusters. We envision a scenario in which almost simultaneously with the assembly of signaling proteins in the microclusters, disassembly mechanisms are triggered to limit the duration of signaling. In this study, we have uncovered the ubiquitylation and endocytosis of signaling complexes as one possible mechanism of downregulation of signaling proteins at the microcluster level.
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ACKNOWLEDGMENTS |
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This research was supported by the Intramural Research Program of the Center for Cancer Research, NCI, NIH. A.G. and M.S.I. received salary support from the Office of Research on Women's Health, Foundation for Advanced Education in the Sciences, NIH.
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FOOTNOTES |
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Published ahead of print on 15 October 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
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