Correspondence to Arthur Weiss: aweiss{at}medicine.ucsf.edu
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Introduction |
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Generation of the ßTCR repertoire is initiated at the most immature stage of thymocyte development (Sebzda et al., 1999). At this early stage of development, thymocytes express neither CD4 nor CD8 coreceptors. Thymocytes that are destined to become
ß T cells stochastically rearrange their TCRß genes. If rearrangement of TCRß is successful (in frame), the TCRß chain is transported together with a nonvariant pre-TCR
chain and the CD3 complex to the cell surface as the pre-TCR complex. Surface expression of the pre-TCR complex induces ligand-independent signals (Irving et al., 1998), which allows for thymocytes to proliferate and up-regulate CD4 and CD8 expression, thereby progressing to the CD4+ CD8+ double-positive (DP) stage of development. At the DP stage of development, thymocytes rearrange their TCR
genes. If the rearrangement of TCR
is successful, low levels of the mature TCRCD3 complex are expressed on the surface of the developing thymocyte. Expression of the TCRCD3 complex is crucial at this stage of development, as signals through the TCR are required for the survival (positive selection) or deletion (negative selection) of DP thymocytes (Sebzda et al., 1999; Love and Chan, 2003). If the TCR expressed by a DP thymocyte cannot bind to self-peptideMHC molecules, the thymocyte fails to receive positively selecting signals and subsequently dies. Conversely, if the TCR interacts too strongly with self-peptideMHC molecules, the cell is potentially autoreactive and is deleted via apoptosis. Thymocytes that express TCRs with an intermediate affinity for peptideMHC down-regulate either CD4 or CD8, thereby progressing to the single-positive (SP) stage of development. Progression to this more phenotypically and functionally mature SP stage is also associated with a 10-fold increase in the level of the TCRCD3 complex to that of mature peripheral T cells. At the SP stage, thymocytes are subjected to further selection and maturation processes before exiting the thymus as mature T cells.
Both positive and negative selection processes are dependent on the strength of signals received through the TCRCD3 complex. Signal strength is dependent not only on the intrinsic affinity of the TCR for peptideMHC molecules but also on the number of receptors that interact with peptideMHC. TCRCD3 expression on DP thymocytes is only 10% of the level observed on SP thymocytes and mature T cells (Finkel et al., 1987; Havran et al., 1987). Previous studies have demonstrated that modulating levels of TCRCD3 expression in developing thymocytes can lead to alterations in positive selection (Ericsson and Teh, 1995; Naramura et al., 1998; Sosinowski et al., 2001), suggesting that tight regulation of surface TCRCD3 levels is required for normal TCR repertoire selection. Therefore, proteins that regulate surface TCRCD3 levels in the thymus are likely to be important determinants of thymocyte development.
Recently, we have shown that Src-like adaptor protein (SLAP) regulates the level of TCRCD3 expression on DP thymocytes (Sosinowski et al., 2001). SLAP was identified in a yeast two-hybrid screen for proteins that interact with the cytoplasmic domain of the Eck receptor protein tyrosine kinase (Pandey et al., 1995). In particular, SLAP is highly homologous with Src family kinases, which include the T lymphocytespecific family members of Lck. Like Src family kinases, SLAP has a unique NH2 terminus that is myristolated, thereby targeting SLAP to cellular membranes (Manes et al., 2000). The NH2 terminus of SLAP is followed by Src homology (SH) 3 and 2 domains, which share 55 and 50% amino acid sequence identity with these domains in Lck, respectively. Unlike Src family kinases, however, SLAP lacks a kinase domain and, instead, contains a unique COOH terminus, whose function remains unclear.
Because SLAP is highly homologous to Lck but lacks a kinase domain, it was postulated that SLAP could negatively regulate Src family kinases by functioning in a dominant negative manner. Overexpression and microinjection studies in nonlymphoid cells have shown that SLAP can inhibit Src-mediated signaling through the platelet-derived growth factor receptor (Roche et al., 1998; Manes et al., 2000). In Jurkat T cells, the transient overexpression of SLAP can inhibit signaling downstream of the TCR as measured by nuclear factor of activated T cells, AP-1, or interleukin 2 transcriptional reporter activity (Sosinowski et al., 2000). Maximum inhibition of nuclear factor of activated T cell activity in Jurkat T cells requires both the SH2 and SH3 domains of SLAP. Altogether, these data suggest that SLAP is an inhibitor of Src family kinases. However, the mechanism by which SLAP inhibits signaling remains unclear, as no differences in overall tyrosine phosphorylation were observed in Jurkat T cells that overexpressed SLAP or in SLAP/ thymocytes (Sosinowski et al., 2000, 2001).
In mice, SLAP protein expression is developmentally restricted and is most highly expressed in DP thymocytes (Sosinowski et al., 2001). Consistent with this restricted pattern of expression, targeted inactivation of the SLAP gene demonstrated that SLAP down-regulates TCRCD3 expression at the DP stage of thymocyte development. In addition to increased TCRCD3 expression, SLAP/ DP thymocytes also display increased levels of CD4, CD5, and CD69. Furthermore, SLAP/ thymocytes display increases in positive selection in the presence of a transgenic TCR. Finally, SLAP deficiency can partially overcome a developmental block at the DP stage and rescue the development of CD4+ SP thymocytes and peripheral T cells in mice that lack ZAP-70 (-associated protein of 70 kD) tyrosine kinase. These alterations in thymocyte development in the absence of SLAP argue that control of surface TCRCD3 levels on DP thymocytes is an important regulatory step in the generation of peripheral T cells. Therefore, we set out to elucidate the mechanism of SLAP-mediated TCRCD3 down-regulation on DP thymocytes. In this study, we show that SLAP-deficient thymocytes have a defect in TCR
chain degradation, which leads to an increased pool of fully assembled TCRCD3 complexes that are capable of recycling back to the cell surface.
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Results |
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Our lab has previously shown that SLAP-mediated TCRCD3 down-regulation on DP thymocytes does not depend on a positively selecting MHC allele (Sosinowski et al., 2001). Therefore, we were interested in studying CD3 recycling in the absence of any TCR ligation. We reasoned that if we could block internalization of the TCRCD3 complex, any new TCRCD3 appearing on the cell surface would be caused by newly synthesized or recycled TCRCD3. To block TCRCD3 internalization, we exploited the observation that TCR is internalized via clathrin-coated pits (Telerman et al., 1987). Hypertonic medium (e.g., 0.45 M sucrose) blocks clathrin-mediated endocytosis by inducing spontaneous clathrin lattice formation in the absence of cell membranes, thereby depleting the cell of clathrin monomers that would be used in vesicle formation (Daukas and Zigmond, 1985; Heuser and Anderson, 1989). Furthermore, hypertonic medium has been shown to inhibit internalization of the TCRCD3 complex (Dallanegra et al., 1988).
To validate the use of hypertonic medium to inhibit TCRCD3 internalization, we first analyzed the uptake of fluorescently labeled transferrin by the transferrin receptor, a process that requires clathrin-mediated endocytosis (Mellman, 1996; Schmid, 1997). Because DP thymocytes do not express the transferrin receptor, we first tested the effect of hypertonic medium on Jurkat T cell lines that stably expressed either SLAP-GFP fusion or GFP alone as a control. Transferrin uptake via the transferrin receptor was completely inhibited by hypertonic medium in both stable cell lines (Fig. 4 A), demonstrating that hypertonic medium blocks clathrin-mediated endocytosis regardless of whether SLAP is present or not. We analyzed the effect of SLAP on CD3 expression while clathrin-mediated endocytosis was blocked in Jurkat T cells. Strikingly, we were able to detect up-regulation of CD3
surface expression over the time course of the experiment in the control cell line. However, up-regulation of CD3
by Jurkat T cells was markedly inhibited in the presence of SLAP (Fig. 4 B).
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Previous studies have reported that prolonged culture of DP thymocytes in the absence of TCRMHC interactions results in up-regulation of the TCRCD3 complex by the stabilization of newly synthesized TCR chains (Bonifacino et al., 1990; Kearse et al., 1995). Indeed, both WT and SLAP/ DP thymocytes up-regulate CD3
when cultured in the absence of 0.45 M sucrose (Fig. 4 E). However, over this time course, the increase in CD3
expression was markedly lower than the increase observed in thymocytes that were incubated in hypertonic medium. In addition, most of the CD3
up-regulation that was observed in hypertonic medium was detected after only 2 h in culture, a time at which little or no CD3
up-regulation had yet occurred in the absence of 0.45 M sucrose. Furthermore, incubation of thymocytes in cycloheximide to inhibit protein synthesis had no effect on the up-regulation of CD3
in hypertonic medium (Fig. 4 D). In contrast, in parallel experiments using thymocytes cultured in media without 0.45 M sucrose, up-regulation of CD3
was sensitive to cycloheximide treatment (unpublished data), demonstrating that cycloheximide effectively inhibited protein synthesis in our cultures.
It is possible that incubation of thymocytes in hypertonic medium has an effect on cell viability. However, Annexin V staining failed to demonstrate a decrease in thymocyte viability for thymocytes incubated in hypertonic medium (unpublished data). In addition, because SLAP protein expression is predominantly restricted to DP thymocytes (Sosinowski et al., 2001), up-regulation of CD3 in hypertonic medium should not be altered in SP thymocytes. Notably, CD3
up-regulation by CD4+ SP thymocytes that were cultured in hypertonic medium was similar regardless of genotype (Fig. 4 D). These data demonstrate that up-regulation of TCRCD3 expression in hypertonic medium can be used to analyze the pool of recycling TCRCD3 complexes in the absence of receptor cross-linking.
The TCR chain is the target of SLAP
We have demonstrated that expression of TCR is increased in SLAP/ thymocytes as a result of the impaired degradation of TCR
. A SLAP-GST fusion has previously been shown to interact with several phosphoproteins, including the TCR
chain (Tang et al., 1999; Sosinowski et al., 2000). Therefore, we postulated that SLAP could bind to TCR
, leading to its subsequent degradation. To study the interaction between SLAP and TCR
, we first examined Jurkat T cells that had been transiently transfected with SLAP-GFP to determine whether SLAP can interact with the endogenous TCR
chain. SLAP-GFP, but not the GFP control, coimmunoprecipitated with TCR
(Fig. 5 A). Furthermore, SLAP-GFP containing a point mutation in the SH2 domain coimmunoprecipitated only weakly with TCR
. The cytoplasmic domain of TCR
is phosphorylated by Lck (Iwashima et al., 1994; van Oers et al., 1996). Therefore, the inhibition of Src family kinase catalytic activity with PP2 caused a loss of basal TCR
phosphorylation in Jurkat T cells (Fig. 5 A). Interestingly, PP2 treatment also caused a corresponding loss in SLAP-GFP coimmunoprecipitation with TCR
. The dependence on Src family kinase activity prompted us to study whether SLAP can interact with TCR
in the Lck-deficient Jurkat T cell line JCaM1. Transiently transfected SLAP-GFP failed to coimmunoprecipitate with TCR
in JCaM1; however, stable reconstitution of Lck back into the JCaM1 cell line resulted in the restoration of basal phospho-TCR
as well as the recovery of SLAP-GFP coimmunoprecipitation with TCR
(Fig. 5 B).
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It has previously been shown that upon transient transfection, SLAP localizes to an intracellular compartment and displays partial colocalization with late endosomes (Sosinowski et al., 2000). Therefore, we predicted that SLAP would colocalize with TCR in an intracellular compartment. Extensive colocalization of SLAP-GFP with endogenous TCR
was observed after transient transfection into Jurkat T cells (Fig. 5 D). Colocalization occurred primarily in an intracellular compartment, as very little SLAP-GFP was detected at the plasma membrane. Interestingly, colocalization requires the SH2 domain of SLAP, as an SH2 point mutation of SLAP-GFP displayed very little colocalization with the TCR
chain. These data suggest that the SH2 domain of SLAP is required for SLAP to interact with phosphorylated TCR
, thus targeting TCR
for degradation.
To investigate the requirement of TCR cytoplasmic domains for the effects of SLAP on TCR expression in vivo, we obtained TCR
-deficient mice that were reconstituted with transgenes encoding either full-length TCR
(TCR
Tg) or a cytoplasmic truncation of TCR
(TCR
D67150; Shores et al., 1994). TCR
D67150 is a deletion of amino acid residues 67150 of TCR
, resulting in the loss of five out of six tyrosines that are normally present in the TCR
cytoplasmic domain. Interestingly, relative to mice expressing TCR
Tg, mice expressing TCR
D67150 displayed increased surface levels of TCRß and CD3
on DP, but not SP, thymocytes (Fig. 5 F and not depicted). Furthermore, mice expressing the truncated form of TCR
displayed increased CD3
up-regulation in hypertonic medium on DP, but not SP, thymocytes (Fig. 5 G), suggesting that the TCRCD3 recycling pool is increased in the absence of the TCR
cytoplasmic domain. The observed increase was not caused by new synthesis of the complex, as cycloheximide had no effect on TCRCD3 recycling in hypertonic medium (unpublished data). Additionally, no significant increase in CD3
expression was observed in thymocytes that were incubated in the absence of 0.45 M sucrose (unpublished data). These data indicate that the cytoplasmic domain of TCR
is required to prevent the accumulation of fully assembled TCRCD3 complexes in the recycling pool of DP thymocytes.
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Discussion |
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In addition to Lck, our studies suggest that regulation of TCRCD3 expression by SLAP requires the cytoplasmic domain of TCR. TCR
-deficient mice that are transgenic for a truncated form of TCR
(TCR
D67150) express increased levels of surface TCRCD3 as well as an increased pool of recycling CD3
on DP, but not SP, thymocytes. It is possible that in the absence of the majority of TCR
cytoplasmic domains, trafficking of the TCRCD3 complex is altered such that internalization and/or recycling of the complex leads to the phenotype that we have observed in TCR
D67150 DP thymocytes via a SLAP-independent mechanism. Notably, the TCR
cytoplasmic domain contains multiple tyrosine-based motifs that could mediate internalization of the TCRCD3 complex. However, a recent study demonstrated that the mutation of all six tyrosines present in the TCR
cytoplasmic domain had no effect on the internalization of the TCRCD3 complex (Szymczak and Vignali, 2005). An additional study has indicated that a similar deletion of the TCR
cytoplasmic domain increases the rate of TCRCD3 internalization (D'Oro et al., 2002). This later study suggests that, if anything, TCRCD3 expression should be decreased in the absence of the TCR
cytoplasmic domain. Nonetheless, we cannot exclude the possibility that altered trafficking of the TCRCD3 complex either contributes to or is responsible for the increased surface TCRCD3 expression and increased pool of recycling CD3
in TCR
D67150 DP thymocytes. However, our findings are also consistent with a role for the phosphorylated TCR
cytoplasmic domain interacting with SLAP, thereby leading to its accelerated degradation.
Previous data indicate that unassembled TCR chains are rapidly degraded in the ER of DP thymocytes (Bonifacino et al., 1990) and that newly synthesized TCR
chains are stabilized by their incorporation into fully assembled TCRCD3 complexes (Kearse et al., 1995). This raised the possibility that SLAP could function to prevent assembly of the TCRCD3 complex. Currently, it is believed that the rate of TCRCD3 assembly is low in DP thymocytes as a result of the instability of the TCR
chain. However, because we failed to detect a difference in expression, synthesis, or degradation of the TCR
chain in the absence of SLAP, we must conclude that SLAP does not regulate assembly of the TCRCD3 complex by the mechanism previously described. In addition, our data clearly demonstrate that SLAP interacts with the phosphorylated TCR
chain, which has been shown to be present only in fully assembled, mature TCRCD3 complexes (Kearse et al., 1993). Therefore, it is unlikely that SLAP plays a direct role in the assembly of TCRCD3. We cannot exclude the possibility that SLAP somehow regulates TCRCD3 assembly through an indirect mechanism that has yet to be described. However, our data suggests that a substantial proportion of TCR
is present mainly in fully assembled TCRCD3 complexes, as evidenced by the large increase in TCR
chain protein expression in WT thymocytes as compared with TCR
/ thymocytes (Fig. 1 A). Together, these data strongly indicate that the degradation of TCR
observed in WT DP thymocytes is predominantly caused by the degradation of TCR
that was derived from fully assembled TCRCD3 complexes.
The TCR has been shown to undergo constitutive internalization in cell lines and T cells, with an estimate of 0.61.2% of TCRs internalized per minute (Liu et al., 2000; Menne et al., 2002). Therefore, subtle modifications to the rate of recycling or internalization could have large effects on the steady-state level of TCR expression on the cell surface. We consistently observed an increase in both the rate as well as the absolute amount of CD3 recycled by SLAP/ DP thymocytes. Notably, the increased amount of CD3
recycled (59% as calculated from Fig. 3 B) is consistent with the 57.5% of TCR
chains that are estimated to be phosphorylated in the thymus (van Oers, N., personal communication). Furthermore, the small difference in CD3
recycling can account for the loss of TCR
that is observed over time in WT but not for SLAP/ thymocytes when incubated in cycloheximide. Therefore, SLAP appears to target only a small proportion of the constitutively recycling TCRCD3 complexes that contain phospho-TCR
. However, this has a substantial effect on the steady-state level of TCRCD3 expression in SLAP-deficient thymocytes.
In contrast to TCR, we were unable to detect a substantial increase in the level of TCR
, TCRß, or CD3
in the absence of SLAP. Likewise, degradation of TCR
, TCRß, and CD3
was not noticeably altered in the absence of SLAP. One possible explanation for these observations is that DP thymocytes typically express a relatively large intracellular pool of unassembled and/or partially assembled TCRCD3 chains, some of which are rapidly degraded (Chen et al., 1988; Lippincott-Schwartz et al., 1988; Bonifacino et al., 1989, 1990). Therefore, the large pools of unassembled TCRCD3 chains could mask any differences in TCRCD3 expression or degradation. Alternatively, our results could also be interpreted to indicate that TCR
separates from the rest of the mature TCRCD3 complex and is independently degraded via a SLAP-dependent mechanism. Separation of the TCRCD3 complex has been previously described (Kishimoto et al., 1995; Ono et al., 1995; Thien et al., 2003; La Gruta et al., 2004). Additional studies will be required to conclusively determine whether the remainder of the recycling TCRCD3 complex is also degraded via a SLAP-dependent mechanism or is retained in an intracellular compartment.
The mechanism by which SLAP targets TCR for degradation has yet to be elucidated. Degradation of TCR
is likely to involve the E3 ubiquitin ligase c-Cbl (Fig. 6). SLAP has been previously reported to interact with the NH2 terminus of c-Cbl (Tang et al., 1999). In addition, c-Cbldeficient mice have a very similar phenotype to SLAP-deficient mice with regard to TCRCD3, CD4, and CD5 up-regulation on DP thymocytes and increases in positive selection (Naramura et al., 1998). Finally, TCR
has been previously shown to be ubiquitinated (Cenciarelli et al., 1992, 1996; Hou et al., 1994), perhaps via c-Cbl (Wang et al., 2001). Therefore, we suspect that the ubiquitin ligase activity of c-Cbl is required to target TCR
for degradation and subsequently prevent the accumulation of recycling TCRCD3 complexes. Indeed, our unpublished results indicate that c-Cbl and SLAP function in the same pathway to regulate TCRCD3 expression on DP thymocytes by targeting the TCR
chain for degradation (unpublished data). Therefore, we suggest the model shown in Fig. 6. Surface TCRCD3 complexes in DP thymocytes are constitutively phosphorylated on TCR
chains by Lck. The TCRCD3 complex is internalized and transported to an intracellular compartment where SLAP binds to phosphorylated TCR
, thus targeting TCR
for ubiquitination and degradation via a c-Cbldependent mechanism. In the absence of TCR
, the remainder of the TCRCD3 complex is either degraded or retained in an intracellular compartment. Conversely, in the absence of SLAP, the TCR
is neither ubiquitinated nor degraded, and the TCRCD3 complex remains intact and continues to recycle back to the plasma membrane.
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Materials and methods |
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Mice
SLAP/ mice have been described previously (Sosinowski et al., 2001) and have been backcrossed to a total of eight generations onto a C57BL/6 background. C57BL/6 mice (Taconic) were used as WT controls. ZAP-70/ mice have been previously described (Kadlecek et al., 1998). TCR/ mice were purchased from the Jackson Laboratory. TCR
Tg and TCR
D67250 were provided by E. Shores (Food and Drug Administration, Bethesda, MD) and have been previously described (Shores et al., 1994).
FACS staining
After washing in PBS, thymocytes were stained with CD4 (RM4-5; eBioscience), CD8 (53-6.7; BD Biosciences), CD3
(145-2C11; eBioscience), and TCR
(H57-197; eBioscience) in FACS buffer (PBS with 1% BSA and 0.01% NaN3) for 30 min at 4°C. For intracellular FACS staining, thymocytes were washed in PBS before fixation in 4% PFA for 20 min at RT. After washing, thymocytes were permeabilized in 0.5% saponin in FACS buffer for 20 min at RT. Thymocytes were stained with the antibodies listed above in 0.5% saponin containing 1% goat serum for 30 min at 4°C. Jurkat T cells were stained with
-CD3
(UCHT1; BD Biosciences) in FACS buffer for 30 min at 4°C. All FACS staining was performed in duplicate.
Internalization and degradation assays
For ligand-induced CD3 internalization, surface CD3
was labeled with biotinylated anti-CD3
antibodies (145-2C11) for 30 min on ice. Thymocytes were washed, resuspended at 5 x 106 ml in primary cell culture medium (RPMI containing 10% FBS, 2 mM glutamine, 50 µM ß-mercaptoethanol, penicillin, and streptomycin), and incubated at 37°C for the indicated time points. Anti-CD3
remaining on the cell surface was detected by staining with PE-conjugated streptavidin (Caltag Laboratories). For degradation assays, thymocytes were cultured at 5 x 106 ml in primary cell culture medium containing 100 µg/ml cycloheximide. At each time point, cells were mixed with 100 µl ice-cold PBS containing 1% BSA and 0.1% NaN3. Cells were maintained on ice for the remainder of the assay until FACS staining.
CD3 recycling
20 x 106 ml of freshly isolated thymocyte single-cell suspensions were cultured at 37°C in primary cell culture media containing 5 µg/ml PE-labeled anti-CD3 (145-2C11). After 30 min in culture, thymocytes were washed twice with PBS. Surface-bound antibody was removed by washing thymocytes twice in ice-cold PBS containing 1% BSA, pH 1.5, followed by immediate neutralization in FACS buffer. These conditions removed >97% of cell surface staining. 5 x 106 ml of thymocytes were incubated at 37°C in primary cell culture for the indicated time points. At each time point, cells were mixed with 100 µl of ice-cold PBS containing 1% BSA and 0.1% NaN3 and were maintained on ice for the remainder of the assay. After all time points had been collected, thymocytes were again washed twice in ice-cold PBS containing 1% BSA, pH 1.5, followed by immediate neutralization in FACS buffer to remove anti-CD3
antibody that was bound to recycled CD3
. Thymocytes were stained for CD4 and 8 and were analyzed by FACS. Recycled CD3
was calculated using the following formula:
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Labeling of recycling pool
Freshly isolated thymocyte single-cell suspensions were cultured at 20 x 106 ml in primary cell culture media containing 100 µg/ml cycloheximide and 5 µg/ml PE-labeled anti-CD3 (145-2C11) for 30 min on ice to fully label surface CD3
. This was followed by a subsequent 60-min incubation either on ice or at 37°C to achieve steady-state labeling of the recycling pool. Cells were washed, stained for CD4 and 8 expression, and analyzed by FACS.
Hypertonic recycling assay
106 ml Jurkat T cells (in RPMI) or 5 x 106 ml of freshly isolated thymocyte single-cell suspensions (in primary cell culture media) were incubated with 0.45 M sucrose. At each time point, cells were mixed with 100 µl of ice-cold PBS containing 1% BSA and 0.1% NaN3. Cells were maintained on ice for the remainder of the assay until FACS staining.
Inhibition of clathrin-mediated endocytosis was measured by the inhibition of transferrin uptake. Jurkat T cells were incubated on ice with Alexa647-labeled transferrin (Molecular Probes) for 20 min to allow for transferrin binding. Excess transferrin was washed off with three washes in ice-cold PBS. Cells were resuspended in RPMI ± 0.45 M sucrose and were incubated either on ice or at 37°C for 10 min. After incubation, cells were transferred into ice-cold PBS containing 1% BSA and 0.1% NaN3 and rested for 1 h on ice. Surface-bound transferrin was competed off by using a 50-fold excess of unlabeled transferrin (Sigma-Aldrich) in FACS buffer for 30 min at RT. Internalized transferrin was calculated by using the following formula:
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Western blotting and immunoprecipitations
For total levels of TCR, CD3
, and TCR
, CD8+ thymocytes were purified by using magnetic cell sorting (Miltenyi Biotec) according to the manufacturer's protocol. Recovered cells were
95% DP, as assessed by FACS. CD8+ thymocytes were lysed at 200 x 106 ml in radioimmunoprecipitation (RIPA) lysis buffer supplemented with protease inhibitors (leupeptin, aprotinin, PMSF, and pepstatin A) for 30 min on ice. Postnuclear supernatants were prepared by centrifuging samples at 16,000 g (4°C) for 30 min. For TCR
, postnuclear supernatants from 50 x 106 cell equivalents were immunoprecipitated with
-TCR
(H-142; Santa Cruz Biotechnology, Inc.) and protein G (GE Healthcare) for 1 h at 4°C. Samples were washed four times with lysis buffer, resuspended in SDS loading buffer, and boiled for 5 min. For CD3
and TCR
, postnuclear supernatants from 5 x 106 cell equivalents were mixed with SDS loading buffer and were boiled. Samples were electrophoresed in a 12.5% SDS-PAGE gel, transferred to Immobilon, and blotted for
-tubulin (B-5-1-2; Sigma-Aldrich), TCR
(H-142), CD3
(M20; Santa Cruz Biotechnology, Inc.), or TCR
(8D3; BD Biosciences). Subsequently, membranes were incubated in secondary antibodies coupled to HRP (GE Healthcare) and were detected by using enhanced chemiluminescence (GE Healthcare). For TCRCD3 degradation experiments, CD8+ thymocytes were cultured in primary cell culture media containing 100 µg/ml cycloheximide. At each time point, thymocytes were lysed in RIPA lysis buffer and were maintained on ice for the remainder of the experiment. Lysates were blotted for
-tubulin, TCR
, CD3
, or TCR
as described above. Western blots were quantitated on an Image Station (Eastman Kodak Co.) using 1D Image Analysis software version 3.5 (Eastman Kodak Co.).
For SLAP/TCR coimmunoprecipitations, cells were transiently transfected with GFP, SLAP-GFP, or SH2-GFP overnight. Cells were washed twice in PBS and were lysed at 50 x 106 ml in RIPA lysis buffer as described above (but supplemented with NaVO4). Postnuclear supernatants from 60 x 106 cells were immunoprecipitated with TCR
(6B10.2; Santa Cruz Biotechnology, Inc.) and protein G as described above. Samples were electrophoresed in a 12.5% SDS-PAGE gel, transferred to Immobilon, and blotted for GFP (JL8; CLONTECH Laboratories, Inc.) or phosphotyrosine (4G10; Upstate Biotechnology) as described for Western blotting. Subsequently, membranes were stripped (30 min at 55°C in 5% SDS, 100 mM ß-mercaptoethanol, and 62.5 mM Tris, pH 6.8) and blotted for TCR
(8D3; BD Biosciences). In some experiments, transfected cells were incubated overnight with 20 µM PP2 (Calbiochem) to inhibit Src family kinase activity.
Metabolic labeling
80 x 106 freshly isolated thymocytes were cultured for 30 min in 4 ml cysteine and methionine-free media (Biofluids) at 37°C. Thymocytes were labeled with 2 mCi of Tran35SLabel (ICN Biomedicals) for 30 min at 37°C. Cells were washed, lysed in RIPA lysis buffer as described above, and precleared for 30 min at 4°C with protein G. Lysates were immunoprecipitated for TCR (H-142), CD3
(M-20), or TCR
(6B10.2), run on an SDS-PAGE gel, and transferred to Immobilon as described above. Half of each immunoprecipitate was used to detect the Tran35SLabel. The other half of the immunoprecipitate was Western blotted (as described above) as a loading control.
Immunofluorescence
Jurkat T cells were transfected with GFP, SLAP-GFP, or SH2-GFP overnight. Transfected cells were washed and allowed to settle onto poly-L-lysinecoated plates. Samples were fixed in 4% PFA (20 min at RT) and were permeabilized with 0.1% Triton X-100 (10 min at RT). Samples were incubated in blocking buffer (PBS with 0.5% BSA, 0.5% milk, and 1% goat serum) for 10 min at RT and were incubated in primary antibody (6B10.2, 1:50) in blocking buffer for 2 h at 37°C. After washing, samples were incubated in secondary antibody (Cy5 goat antimouse IgG; Jackson ImmunoResearch Laboratories) in blocking buffer for 20 min at RT. Samples were washed, coverslipped in gel/mount (Biomeda), and visualized on a turnkey inverted digital microscopy system (Marianas; Intelligent Imaging) that was built around an inverted microscope (Axiovert 200M; Carl Zeiss MicroImaging, Inc.) using a plan-Neofluar 40x oil immersion objective (NA 1.3; Carl Zeiss MicroImaging, Inc.). Images were collected at RT with a CCD SensiCam (PCD; Cooke Corp.) using SlideBook software (Intelligent Imaging), were deconvolved using a constrained iterative algorithm (SlideBook), and were exported as TIFF files.
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Acknowledgments |
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Submitted: 31 January 2005
Accepted: 15 June 2005
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References |
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