Localization of Src Homology 2 Domain-Containing Phosphatase 1 (SHP-1) to Lipid Rafts in T Lymphocytes: Functional Implications and a Role f
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免疫学杂志 2005年第5期
Abstract
The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) has previously been shown to be a negative regulator of signaling mediated via the TCR. A growing body of evidence indicates that the regulated localization of proteins within certain membrane subdomains, referred to as lipid rafts, is important for the successful transduction of signaling events downstream of the TCR. However, considerably less is known about the localization of negative regulators during these lipid raft-dependent signaling events. In this study we have investigated the subcellular localization of SHP-1 and its role in regulation of TCR-mediated signaling. Our studies demonstrate that in a murine T cell hybridoma as well as in primary murine thymocytes, a fraction of SHP-1 localizes to the lipid rafts, both basally and after TCR stimulation. Interestingly, although SHP-1 localized in the nonraft fractions is tyrosine phosphorylated, the SHP-1 isolated from the lipid rafts lacks the TCR-induced tyrosine phosphorylation, suggesting physical and/or functional differences between these two subpopulations. We identify a requirement for the C-terminal residues of SHP-1 in optimal localization to the lipid rafts. Although expression of SHP-1 that localizes to lipid rafts potently inhibits TCR-mediated early signaling events and IL-2 production, the expression of lipid raft-excluded SHP-1 mutants fails to elicit any of the inhibitory effects. Taken together these studies reveal a key role for lipid raft localization of SHP-1 in mediating the inhibitory effects on T cell signaling events.
Introduction
Signaling through the TCR is an event that is critical for both T cell development and mature T cell function. Activation of T cells through the TCR induces tyrosine phosphorylation of numerous intracellular proteins and evokes a signaling cascade that, depending on the differentiation state of the T lymphocyte, promotes proliferation, differentiation, or cell death. One of the initial events that occurs after stimulation through the TCR is the rapid tyrosine phosphorylation of ITAM motifs (ITAMs) residing in the cytoplasmic domains of the CD3 chains; this phosphorylation is mediated by the Src family protein tyrosine kinases Lck and Fyn (reviewed in Refs. 1, 2, 3). Simultaneous engagement of the coreceptor CD4 or CD8 amplifies the signal by increasing the amount of activated Lck in close proximity to the TCR (reviewed in Refs. 1 and 3). ZAP70, a Syk/ZAP70 family nontransmembrane protein tyrosine kinase, binds via its Src homology 2 (SH2)3 domains to the phosphorylated ITAMs of CD3 and is subsequently activated by Lck and/or Fyn (reviewed in Refs. 2 and 3). ZAP70 then transmits and amplifies downstream signals by facilitating the tyrosine phosphorylation of proteins such as linker for activation of T cells (LAT), phospholipase C1, Vav, and SH2 domain-containing leukocyte protein of 76 kDa (reviewed in Refs. 2, 3, 4, 5, 6, 7).
During T cell signaling, a critical role has been recognized for one type of membrane subdomain, often referred to as lipid rafts (reviewed in Refs. 8, 9, 10, 11). Enrichment in cholesterol and glycosphingolipids allows these microdomains a tightly packed, highly ordered structure within the more fluid plasma membrane (reviewed in Refs. 12, 13, 14). Lipid rafts have been defined biochemically as being insoluble in nonionic detergent at 4°C (reviewed in Refs. 15, 16, 17). In addition to the biochemical studies, various microscopic techniques have allowed visualization of lipid rafts and provided further evidence that some molecules are constitutively anchored in lipid rafts, whereas others undergo a regulated traffic to the lipid rafts (18, 19, 20, 21, 22, 23, 24, 25 ; reviewed in Refs. 11 , 26 , and 27). It has recently been shown that a number of proteins involved in signaling through the TCR localize to the lipid rafts either constitutively, such as CD4, CD8, Lck, Fyn, LAT, phosphoprotein associated with GEMS/Csk-binding protein, and LIME (20, 28, 29, 30, 31, 32, 33, 34, 35), or in a stimulus-dependent manner, such as ZAP70, Shc, SH2 domain-containing leukocyte protein of 76 kDa, Grb2-related adaptor downstream of Shc, and phospholipase C1 (29, 35, 36). Other proteins, such as Csk and the transmembrane phosphatase CD45 (30, 35, 37, 38, 39), appear to be excluded from the lipid rafts after activation. The importance of lipid rafts in T cell signaling was also emphasized by several studies illustrating that disruption of lipid rafts and/or displacement of signaling molecules from the lipid rafts abolishes TCR-mediated signaling events (29, 36, 40, 41, 42, 43, 44).
Although much is known about the initiation of TCR-mediated signaling events, relatively less is known about the mechanism(s) by which signaling is negatively controlled or terminated (reviewed in Refs. 45, 46, 47). The dephosphorylation of cellular phosphoproteins is considered to be an important part of this process, because the rapid rise in intracellular tyrosine phosphorylation that occurs upon T cell stimulation is transient, decreasing within minutes of the initial response. Given that tyrosine phosphorylation declines rapidly after the initial stimulation response, protein tyrosine phosphatases might reside or be recruited to the lipid rafts to rapidly terminate signaling. Numerous studies conducted in primary thymocytes, T cell lines, and T cell hybridomas have revealed the protein tyrosine phosphatase SH2 domain-containing phosphatase 1 (SHP-1) as a negative regulator of TCR-mediated signaling (reviewed in Ref. 45, 46 , and 48). Based on studies using T lymphocytes derived from the SHP-1-negative motheaten mice (49, 50) and cell lines overexpressing dominant negative mutants of SHP-1 (51, 52), SHP-1 does not seem to affect basal tyrosine phosphorylation and therefore probably exists basally in an inactive state. After TCR stimulation, SHP-1 is thought to become activated, potentially by engagement of its SH2 domains with an as yet unidentified partner and is then able to act on potential targets, such as Lck (49, 53), ZAP70 (51, 54), Vav (55), PI3K (56), and potentially the CD3 chains (57). Because SHP-1 has been shown to be involved in the negative regulation of signaling events initiated through the TCR, and many of its proposed substrates localize to the lipid rafts, we investigated whether SHP-1 localizes to lipid rafts, and how such localization may be functionally important. Interestingly, in two recent studies, SHP-1 appeared to not localize by itself to the lipid rafts. However, targeting SHP-1 to the lipid rafts inhibited TCR-mediated signaling events, indicating that a role for SHP-1 may indeed exist within the lipid rafts (58, 59).
In this study we demonstrate that in a murine T cell hybridoma and in primary murine thymocytes, a fraction of SHP-1 localized to the lipid rafts. Several lines of investigation suggest that localization of SHP-1 to the lipid rafts is mediated via its C terminus and is essential for the negative regulatory role of SHP-1 in TCR-mediated signaling.
Materials and Methods
Cell culture, generation of cell lines, and primary thymocytes
Parental BYDP (60) and BYDP transfectants stably expressing hemagglutinin (HA)-tagged SHP-1 were grown in RPMI 1640 medium supplemented with 10% FBS, 5 x 10–5 M 2-ME, 2 mM L-glutamine, 10 U/ml penicillin, and 10 μg/ml streptomycin (complete medium). Cell lines expressing HA-tagged SHP-1 variants were maintained under the selection of 1 mg/ml G418.
Stable cells lines were generated by cotransfection of parental BYDP cells with pEBB vectors (61) encoding HA-tagged SHP-1 full-length or mutants and pMH-neo (62), followed by selection for G418 resistance (2 mg/ml). Mutants of SHP-1 were generated via PCR-based mutagenesis. The C terminus of SHP-1 was deleted by replacing the codon for the amino acid at position 527 with a stop codon. The phosphoprotein binding capability of the SHP-1 SH2 domains was disrupted by replacing the codons for conserved arginine residues at positions 30 and 136 with codons for lysine. Two to nine independent clones were isolated for each construct based on expression of the fusion protein.
Murine thymi were obtained from 4- to 8-wk-old C3HeB/FeJLe mice, and single cell suspensions were prepared as described previously (49). Isolated thymocytes were rested for 7 h at 37°C in complete medium (2 x 106 cells/ml) before stimulation.
T cell stimulation and lipid raft isolation
BYDP cells or primary murine thymocytes (6.5 x 107/time point) were used and stimulated as described previously (63). Briefly, cells were costimulated through TCR/CD3 plus CD4 using 1 μg/ml 145-2c11 (Southern Biotechnology Associates) in combination with either 0.5 μg/ml Leu3a anti-human CD4 (BD Biosciences) for BYDP or 1 μg/ml L3T4 anti-mouse CD4 (BD Pharmingen and Southern Biotechnology Associates) for murine thymocytes, followed by secondary Ab cross-linking via the addition of 10 μg/ml goat anti-mouse Ig (Southern Biotechnology Associates). After incubation at 37°C for the indicated times, cells were washed twice in cold PBS containing 1 mM Na3VO4, and lysed at 4°C in 1 ml of TNE lysis buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 5 mM EDTA) containing 0.5% Triton X-100, phosphatase inhibitors (5 mM NaF, 1 mM Na3VO4, and 30 mM -glycerophosphate), and protease inhibitors (10 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml antipain, and 20 μg/ml PMSF). Cell lysates were mixed with an equal volume of 80% sucrose solution. The resultant 40% sucrose solutions containing the lysates were overlaid with 2 ml of 30% sucrose solution and 1 ml of 5% sucrose solution. All sucrose solutions were made up in TNE buffer, maintained at 4°C, and freshly supplemented with phosphatase inhibitors. After ultracentrifugation in an SW55Ti rotor at 200,000 x g for 18 h at 4°C, 11 equal fractions were harvested top to bottom from each gradient. Aliquots of each fraction harvested from sucrose gradients were analyzed by 8% SDS-PAGE and subsequent immunoblotting on Immobilon-P polyvinylidene difluoride membrane (Millipore). Based on the presence of the lipid rafts marker LAT (28) and the lipid rafts-excluded transmembrane protein CD45 (35, 37, 38), fractions 2–4 and 9–10 were identified and subsequently combined as lipid rafts and detergent-soluble fractions, respectively. All pairs of lipid rafts and detergent-soluble fractions presented in the figures were run on the same gel and exposed for the same length of time.
Immunoprecipitation and immunoblotting
Equal aliquots of the combined lipid rafts fractions and detergent-soluble fractions were used for additional analyses (3.75% for analyses of whole fractions and 37.5% for immunoprecipitations). For TCR/CD3 complex precipitations from unfractionated lysates, 107 cells were stimulated by Ab cross-linking, followed by lysis in TNE buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 5 mM EDTA) including protease and phosphatase inhibitors and containing 0.5% Triton X-100 for ZAP70 coprecipitation or 1% Nonidet P-40 for CD3-chain precipitation. Lysates were cleared of nuclear debris by ultracentrifugation at 100,000 x g in a TLA-45 rotor for 20 min at 4°C. Immunoprecipitations were performed as described previously (52, 63) using polyclonal rabbit anti-SHP-1 Abs, monoclonal mouse anti-CD3 IgG1 (Santa Cruz Biotechnology), or polyclonal rabbit anti-LAT Abs (Upstate Biotechnology). Each immunoprecipitation was performed using 2 μg of Abs, because this was the amount determined to effectively clear the lysates. Immune complexes were collected for 1 h at 4°C by addition of 50 μl of protein A-Sepharose beads (Amersham Biosciences) for anti-SHP-1 and anti-LAT immunoprecipitations, or 50 μl of protein G-Sepharose beads (Amersham Biosciences) precoated with goat anti-mouse Abs (Southern Biotechnology Associates) for anti-CD3 immunoprecipitations, followed by five washes with lysis buffer containing phosphatase inhibitors. Immunoprecipitates were resolved by SDS-PAGE and subsequent immunoblotting on Immobilon-P polyvinylidene difluoride membrane (Millipore).
Immunoblottings were performed as described previously (52, 63) using the following primary Abs: mouse anti-phosphotyrosine mAb 4G10 (0.5 μg/ml), polyclonal rabbit anti-LAT Abs (1 μg/ml; Upstate Biotechnology), mouse anti-CD45 mAb (0.5 μg/ml), mouse anti-Lck mAb (1/500), mouse anti-ZAP70 mAb (0.5 μg/ml; BD Transduction Laboratories), and mouse anti-CD3 mAb (0.2 μg/ml; Santa Cruz Biotechnology). SHP-1 immunoblottings were performed using mouse anti-SHP-1 mAb clone 1SH01 (1.33 μg/ml; Neomarkers), with the following exceptions: anti-SHP-1 mAb clone 52 (0.5 μg/ml; BD Transduction Laboratories) and polyclonal rabbit anti-SHP-1 Abs (1 μg/ml; Upstate Biotechnology) were used for comparative SHP-1 Ab detection studies, as indicated in the text. Immunoblots were incubated with the appropriate HRP-conjugated secondary Abs (anti-mouse or anti-rabbit, 1/7500 dilution; Amersham Biosciences) before visualization using ECL.
Pervanadate treatment
A 40 mM stock solution of pervanadate was freshly prepared for each experiment. Equimolar amounts of Na3VO4 and H2O2 were combined and incubated for 20 min at room temperature, followed by addition of an equal volume of 100 mM Tris (pH 7.6), 100 mM HEPES (pH 7.4) containing 4 μg of catalase, and incubation for 30 min at room temperature. Pervanadate treatment was performed by incubating BYDP cells for 5 min at room temperature with a final concentration of 5 mM pervanadate. When pervanadate treatment was used in combination with stimulation via Ab cross-linking, cells were stimulated before treatment with pervanadate.
IL-2 ELISA
Cells (5 x 104) were seeded in 200 μl of complete medium/well of a flat-bottom, 96-well plate coated with the indicated concentrations of the anti-CD3 mAb, 145-2c11 (Southern Biotechnology Associates). As a positive control, PMA (5 ng/ml) and ionomycin (1 μM) were added to cells in uncoated wells. After incubation at 37°C for 24 h, 125 μl of supernatant was harvested from each well. Supernatants (50 μl) were diluted 1/1 with assay diluent (PBS/10% FBS, pH 7.0) and analyzed for IL-2 by ELISA (Mouse IL-2 OptEIA ELISA Set and TMB Substrate Reagent Set; BD Biosciences; used as described by manufacturer). Known concentrations of rIL-2 were used as standards.
Results
SHP-1 localizes to lipid rafts
To determine the basal subcellular localization of SHP-1 in T lymphocytes and whether this localization changes upon stimulation through the TCR, we made use of the BYDP cell line, a murine T cell hybridoma line that mimics various aspects of thymocyte signaling (60, 64) and has previously been used to study SHP-1 and TCR signaling (52, 63). BYDP cells were stimulated via Ab cross-linking (anti-CD3/anti-CD4), followed by isolation of the lipid rafts and detergent-soluble fractions. Overall, the protein phosphotyrosine content in both lipid rafts and detergent-soluble fractions typically peaked after 10 min of stimulation and declined by 20–30 min of stimulation (Fig. 1, top panels). This pattern of tyrosyl phosphorylation of cellular proteins was consistent with previous data (52, 60, 63) and reflected an appropriate stimulation of the cells. Additionally, as observed previously (29, 35), different sets of phosphoproteins were associated with the lipid rafts and the detergent-soluble fractions.
FIGURE 1. SHP-1 localizes to lipid rafts. BYDP cells (6.5 x 107) were stimulated by Ab cross-linking for the indicated times, lysed in 0.5% Triton X-100 lysis buffer, and fractionated via a sucrose step gradient. Combined lipid rafts and detergent-soluble fractions were separated by 8% SDS-PAGE, followed by anti-phosphotyrosine immunoblotting. The blot was stripped and reprobed for the presence of SHP-1, LAT, Lck, and CD45.
Analysis for SHP-1 revealed that a fraction of SHP-1 basally localized to the lipid rafts of BYDP cells (Fig. 1). Band densitometry of a number of immunoblots revealed that 20–30% of total cellular SHP-1 was present in the lipid rafts. However, due to the difficulty in accurately quantitating the protein amounts in lipid rafts and nonlipid rafts, we will from hereon only state that a fraction of SHP-1 localized to lipid rafts. Although the amount of SHP-1 localizing to the lipid rafts was increased in some experiments after anti-CD3/anti-CD4 stimulation, this was not a consistent observation. The purity of the lipid rafts fractions was evaluated by blotting for proteins known to reside in or be excluded from the lipid rafts. Consistent with previous data, 50% of total cellular LAT (28) and 50% of total cellular Lck (31, 32) were localized to the lipid rafts. In contrast, CD45 was absent from the lipid rafts fractions, as previously reported (35, 37, 38), whereas it was readily detectable in the detergent-soluble fractions (Fig. 1).
SHP-1 is hypotyrosyl phosphorylated in lipid rafts
We have previously shown that SHP-1 becomes tyrosyl phosphorylated within its C terminus upon stimulation through TCR/CD3 plus CD4 (63). Mapping of the phosphorylation sites as well as analyses of primary thymocytes and several T cell lines have indicated that this phosphorylation is Lck dependent (63). We examined the tyrosyl phosphorylation status of SHP-1 in the lipid rafts and/or detergent-soluble fractions. Interestingly, SHP-1 was not phosphorylated on tyrosine when immunoprecipitated from the lipid rafts, but was phosphorylated in the detergent-soluble fractions of BYDP cells stimulated via TCR/CD3 plus CD4 (Fig. 2A). Because activated Lck localizes to the lipid rafts (65), and our previous data suggested that Lck is the major tyrosine kinase phosphorylating SHP-1 upon TCR/CD3 plus CD4 stimulation (63), the lack of phosphorylation on lipid raft-associated SHP-1 was surprising. To test whether this reflects a general phenomenon, the experiment was repeated in primary cells. Consistent with our observation in BYDP cells, fractionation of murine thymocytes revealed a hypophosphorylation of lipid raft-associated SHP-1 (Fig. 2B). The observed lack of tyrosyl phosphorylation of SHP-1 cannot be attributed to a general deficiency in protein tyrosyl phosphorylation in the lipid rafts, because a number of phosphoproteins are detectable in the lipid raft fractions upon TCR/CD3 plus CD4 stimulation (Fig. 1, top left panel). Hypophosphorylation of SHP-1 isolated from the lipid raft fractions compared with SHP-1 isolated from the detergent-soluble fractions suggests that there may be functional and/or physical differences between these two subpopulations of SHP-1.
FIGURE 2. SHP-1 is hypo-tyrosyl phosphorylated in lipid rafts of BYDP cells and primary murine thymocytes. BYDP cells (6.5 x 107) (A) or primary murine thymocytes (B) were stimulated by Ab cross-linking for the indicated times. 0.5% Triton X-100 cell lysates were fractionated via a sucrose step gradient. SHP-1 was immunoprecipitated from combined lipid rafts and detergent-soluble fractions, followed by 8% SDS-PAGE and immunoblotting for phosphotyrosyl content. The immunoblots were stripped and reprobed for SHP-1. *, The 70-kDa phosphoprotein migrating above SHP-1 in the detergent-soluble fractions is ZAP70 and is due to the presence of the Abs used for stimulation, rather than a direct interaction with SHP-1 (V. C. J. Fawcett and U. Lorenz, unpublished observation; refer to Results for further explanation).
The 70-kDa phosphoprotein band migrating above SHP-1 in the detergent-soluble fractions isolated from BYDP cells (Fig. 2, top right panel) was identified by immunoblotting as ZAP70 (data not shown). Additional studies suggested that the coprecipitation of ZAP70 is not due to a direct interaction with SHP-1, but results from an indirect interaction via the stimulating Abs to the TCR/CD3 complex (V. C. J. Fawcett and U. Lorenz, unpublished observation). Consistent with published reports, ZAP70 was primarily detected in the detergent-soluble fractions, where the majority of the TCR complexes are found after stimulation via TCR/CD3 Ab cross-linking (66).
Lipid raft-localized SHP-1 can be tyrosyl phosphorylated
One possible explanation for the hypophosphorylation of lipid raft-localized SHP-1 is that SHP-1 cannot be tyrosyl phosphorylated within the lipid rafts. This could be due to a lack of kinases within the lipid rafts or to SHP-1 not being accessible to the available kinases. Moreover, it would imply that there is very limited movement of SHP-1 into or out of the lipid rafts. To determine whether tyrosine phosphorylation of SHP-1 can be achieved at all within the lipid raft environment, we used the tyrosine phosphatase inhibitor pervanadate to achieve maximal, albeit unregulated, tyrosine phosphorylation (67). Treatment of BYDP cells with pervanadate resulted in a rapid and robust tyrosine phosphorylation of cellular proteins (Fig. 3A). An even greater amount of tyrosine phosphorylation was achieved by stimulating the cells via Ab cross-linking (anti-CD3/anti-CD4) before pervanadate treatment, indicating that the ideal localization of either the kinases or the substrates is enhanced by and/or dependent on TCR stimulation. Treatment of BYDP cells with pervanadate resulted in an intense tyrosine phosphorylation of SHP-1 in both the lipid rafts and detergent-soluble fractions (Fig. 3B). Stimulating the cells through TCR/CD3 plus CD4 before pervanadate treatment further augmented the tyrosine phosphorylation of SHP-1 in both the lipid rafts and detergent-soluble fractions (Fig. 3B). Based on these data, we conclude that the C terminus of SHP-1 is accessible to and capable of being tyrosyl phosphorylated within the lipid rafts. Thus, the hypophosphorylation of SHP-1 within the lipid rafts compared with the detergent-soluble fractions is most likely attributable to a heightened dephosphorylation of SHP-1 within the lipid rafts, either via autodephosphorylation or through the action of another phosphatase.
FIGURE 3. SHP-1 is tyrosyl phosphorylated in the lipid rafts after treatment of cells with pervanadate. BYDP cells (6.5 x 107) were treated with 5 mM pervanadate alone or after stimulation by Ab cross-linking. Triton X-100 (0.5%) cell lysates were fractionated via a sucrose step gradient. A, Combined lipid rafts and detergent-soluble fractions were separated by 8% SDS-PAGE and subjected to anti-phosphotyrosine immunoblotting. B, SHP-1 was immunoprecipitated from combined lipid rafts and detergent-soluble fractions, followed by 8% SDS-PAGE and immunoblotting for phosphotyrosyl content. The immunoblots were stripped and reprobed for SHP-1. *, The 70-kDa phosphoprotein migrating above SHP-1 is ZAP70.
Differential Ab detection of lipid rafts-localized SHP-1
During the course of our studies, we made an interesting discovery regarding the C-terminal domain of SHP-1. Compared with anti-SHP-1 polyclonal Abs, an mAb clone 52 (directed against the C terminus of SHP-1; commercially available from BD Transduction Laboratories) displayed a diminished recognition of the highly phosphorylated form of SHP-1 that exists after pervanadate treatment (Fig. 4A, compare middle and bottom panels, lane 4). Additionally, we found that this mAb failed to recognize SHP-1 isolated from the lipid rafts, whereas it readily detected SHP-1 isolated from the detergent-soluble fractions (Fig. 4B, top panels). We were able to detect SHP-1 in the lipid rafts fractions using polyclonal anti-SHP-1 Abs (Fig. 4B, bottom left panel). Notably, there was no difference between the monoclonal and the polyclonal anti-SHP-1 Abs in detecting not hyperphosphorylated SHP-1 isolated from the detergent-soluble fractions (Fig. 4A, lanes 1–3, and Fig. 4B, right panels), indicating that the discrepancy between these Abs is not simply due to a decreased sensitivity of mAb clone 52. This mAb, which recognizes a C-terminal epitope in SHP-1, fails to detect highly phosphorylated SHP-1, most likely because the epitope is masked by the tyrosyl phosphorylation in the C terminus. Because the lipid raft-associated SHP-1 is hypophosphorylated, we hypothesized that this subpopulation of SHP-1 may possess a different posttranslational modification in the C-terminal region, which interferes with recognition by the mAb, and that such a modification could be involved in the localization of SHP-1 to the lipid rafts and/or its function within the lipid rafts.
FIGURE 4. An mAb raised against the C terminus of SHP-1 fails to recognize SHP-1 when either hyperphosphorylated or lipid raft-localized. A, BYDP cells (6.5 x 107) were stimulated by Ab cross-linking and/or 5 mM pervanadate treatment as indicated. Triton X-100 (0.5%) cell lysates were fractionated via a sucrose step gradient, and SHP-1 was immunoprecipitated from the combined detergent-soluble fractions, followed by 8% SDS-PAGE and immunoblotting for phosphotyrosine. *, ZAP70. The immunoblot was stripped and reprobed for SHP-1 using an mAb (clone 52) directed against the C terminus of SHP-1. The immunoblot was stripped again and reprobed for SHP-1 using rabbit polyclonal Abs against SHP-1. B, BYDP cells (6.5 x 107) were stimulated by Ab cross-linking for the indicated times, lysed in 0.5% Triton X-100 lysis buffer, and fractionated via a sucrose step gradient. SHP-1 was immunoprecipitated from the combined lipid rafts and detergent-soluble fractions. Immunoprecipitates were resolved by 8% SDS-PAGE and subjected to immunoblotting for SHP-1 using an mAb (clone 52) directed against the C terminus of SHP-1. The immunoblot was stripped and reprobed for SHP-1 using rabbit polyclonal Abs against SHP-1.
Subcellular localization of mutants of SHP-1
Unlike other lipid raft-localized proteins, such as LAT and Lck, SHP-1 contains no known lipid raft localization sequences. We therefore asked which region/domain(s) of SHP-1 was essential for its localization to lipid rafts by analyzing the subcellular localization of mutants of SHP-1 in BYDP cells. Based on the above observations, we considered a role for the C terminus of SHP-1 in mediating localization to lipid rafts. We generated N-terminally HA-tagged versions of full-length SHP-1 (denoted HA-SHP-1) and a C-terminal deletion mutant (denoted HA-SHP-1C), which lacks the last 68 aa of SHP-1, including the C-terminal tyrosine phosphorylation sites, but does not affect the catalytic domain (see schematic in Fig. 5A).
FIGURE 5. Deletion of the C terminus of SHP-1 impairs localization to the lipid rafts. A, Schematic representation of the versions of HA-tagged full-length and mutant SHP-1 introduced into BYDP cells. B, BYDP cells (6.5 x 107) stably expressing the indicated HA-tagged SHP-1 variants were stimulated by Ab cross-linking for the indicated times, lysed in 0.5% Triton X-100 lysis buffer, and fractionated via a sucrose step gradient. Combined lipid rafts and detergent-soluble fractions were separated by 8% SDS-PAGE and analyzed for the presence of SHP-1 by immunoblotting. Two representative independent clones are shown for each HA-tagged SHP-1 variant.
When HA-SHP-1 was stably expressed in BYDP cells, we found that the relative amounts of total cellular HA-SHP-1 that localized to the lipid rafts and detergent-soluble fractions were comparable to those of endogenous SHP-1, indicating that the HA tag itself did not affect subcellular localization of SHP-1 (Fig. 5B, top left panels). Examination of the subcellular localization of HA-SHP-1C showed that this protein poorly localized to the lipid rafts compared with endogenous SHP-1 or HA-SHP-1 (Fig. 5B, bottom left panels), suggesting that the C terminus of SHP-1 is involved in mediating localization to the lipid rafts. As a control and to test the possible involvement of the SH2 domains of SHP-1 in lipid raft localization, we also generated SHP-1 with point mutations within the SH2 domains, denoted HA-SHP-1RK30/136. The subcellular localization of HA-SHP-1RK30/136 was comparable to that of endogenous SHP-1 (Fig. 5B, top right panels), indicating that the localization of SHP-1 to the lipid rafts is not dependent on functional SH2 domains. However, as observed for the HA-SHP-1C mutant, deletion of the C-terminal 68 aa from the SH2 mutant (denoted HA-SHP-1RK30/136/C) impaired its ability to localize to lipid rafts (Fig. 5B, bottom right panels). These findings implicate a role for the C terminus in mediating the localization of SHP-1 to the lipid rafts and raise the possibility that the C terminus of SHP-1 might possess a novel lipid raft-targeting modification.
Expression of SHP-1and SHP-1RK30/136, but not SHP-1C, affects signaling events initiated through the TCR
We next evaluated the functional consequences of expressing SHP-1 mutants in BYDP cells. It has been previously reported that targeting SHP-1 to the lipid rafts inhibits TCR-mediated signaling events (58, 59). Because the expression of HA-SHP-1 or HA-SHP-1RK30/136 in BYDP cells results in increased representation of SHP-1 within the lipid rafts without artificial targeting to the lipid rafts (Fig. 5B), we asked whether functional consequences would ensue.
We first compared the phosphoprotein profiles of lipid raft-associated and detergent-soluble proteins in the lysates of parental BYDP cells to those expressing HA-SHP-1, HA-SHP-1C, or HA-SHP-1RK30/136 upon TCR/CD3 plus CD4 stimulation. Interestingly, the expression of the poorly lipid raft-localized HA-SHP-1C mutant had no effect on overall tyrosine phosphorylation, whereas expression of HA-SHP-1 and HA-SHP-1RK30/136 resulted in several alterations in the overall phosphoprotein patterns in both the lipid rafts and the detergent-soluble fractions (Fig. 6A, as indicated by arrows). One of the most affected phosphoproteins was in the 35- to 40-kDa range, where a prominent inducible phosphoprotein band was lost from both the lipid rafts and the detergent-soluble fractions in cells expressing HA-SHP-1 or HA-SHP-1RK30/136 (Fig. 6A).
FIGURE 6. Expression of HA-SHP-1 or HA-SHP-1RK30/136, but not HA-SHP-1C, results in the loss of LAT tyrosyl phosphorylation. Parental or HA-tagged SHP-1 variants expressing BYDP cells (6.5 x 107) were stimulated by Ab cross-linking for the indicated times, lysed in 0.5% Triton X-100 lysis buffer, and fractionated via a sucrose step gradient. A, Combined lipid rafts and detergent-soluble fractions were separated by 8% SDS-PAGE and subjected to anti-phosphotyrosine immunoblotting. B, LAT was immunoprecipitated from combined lipid rafts and detergent-soluble fractions. Immunoprecipitates were resolved by 8% SDS-PAGE, followed by immunoblotting for phosphotyrosine and LAT. The SHP-1 expression levels of these representative clones are shown in the bottom panels of Fig. 5B (labeled clone 2).
Because LAT is one of the major proteins phosphorylated in response to TCR stimulation, we hypothesized that this 35- to 40-kDa phosphoprotein band might be LAT. Although phosphorylated LAT is detectable in both the lipid rafts and detergent-soluble fractions after TCR stimulation, previous studies have determined that its phosphorylation takes place within the lipid rafts (28, 68). Therefore, a loss of LAT phosphorylation in cells expressing HA-SHP-1 and HA-SHP-1RK30/136, but not in cells expressing HA-SHP-1C in which SHP-1 is comparatively underrepresented in the lipid rafts, would suggest that SHP-1 expression elicits an inhibitory effect only when localized to lipid rafts. LAT immunoprecipitated from cells expressing HA-SHP-1 or HA-SHP-1RK30/136 displayed a complete loss of inducible tyrosine phosphorylation after TCR/CD3 plus CD4 stimulation, whereas it was readily detectable in parental BYDP (Fig. 6B). Interestingly, tyrosine phosphorylation of LAT immunoprecipitated from cells expressing SHP-1C was comparable to that observed in parental BYDP cells (Fig. 6B). This finding indicates that the inhibitory effect on LAT phosphorylation requires localization of SHP-1 to lipid rafts.
Phosphorylation of LAT requires ZAP70 recruitment to the TCR/CD3 complex. To examine whether this upstream signaling event was also affected in cells expressing HA-SHP-1 or SHP-1RK30/136, we took advantage of our prior observation that ZAP70 coprecipitates with the anti-CD3/anti-CD4 stimulating Abs from BYDP cell lysates (Figs. 2A, 3B, and 4A). Analyses of cells expressing HA-SHP-1 or HA-SHP-1RK30/136 revealed no detectable association between ZAP70 and the TCR complex (Fig. 7A). In contrast, stimulation of parental BYDP and cells expressing HA-SHP-1C readily induced association of ZAP70 with the TCR complex, an event that typically was first detectable at 5–10 min of TCR/CD3 plus CD4 stimulation, with a peak at 15–20 min of stimulation. As expected, little or no basal association of ZAP70 with the TCR/CD3 complex was observed (Fig. 7A). These results indicate that the expression of HA-SHP-1 or HA-SHP-1RK30/136 in BYDP cells inhibits the phosphorylation of LAT by interfering with ZAP70 association with the TCR complex, whereas the expression of HA-SHP-1C fails to elicit these effects, most likely due to its inefficient localization to lipid rafts.
FIGURE 7. Expression of HA-SHP-1 or HA-SHP-1RK30/136, but not HA-SHP-1C, results in a loss of ZAP70 recruitment to the TCR and loss of phosphorylation of the CD3-chains. Parental or HA-tagged SHP-1 variants expressing BYDP cells (107) were stimulated by Ab cross-linking for the indicated times and lysed in 0.5% Triton X-100 lysis buffer (A) or 1% Nonidet P-40 lysis buffer (B). A, The TCR/CD3 complex was precipitated from the cleared lysates. Precipitates were subjected to 8% SDS-PAGE and analyzed for the coprecipitation of ZAP70 by immunoblotting (top panels). Five percent of the cleared lysates were separated by 8% SDS-PAGE and analyzed for ZAP70 and SHP-1 expression (bottom panels). B, CD3 was immunoprecipitated from the cleared lysates. Immunoprecipitates were subjected to 10% SDS-PAGE and analyzed for phosphotyrosyl content by immunoblotting. Immunoblots were stripped and reprobed for CD3 (top panels). Five percent of the cleared lysates were separated by 8% SDS-PAGE and analyzed for SHP-1 expression (bottom panel).
One potential explanation for the lack of ZAP70 association with the TCR complex in cells expressing HA-SHP-1 and HA-SHP-1RK30/136 is that increased representation of SHP-1 in the lipid rafts results in a loss of phosphorylation of the CD3 ITAMs, thereby preventing recruitment of ZAP70. We therefore compared the phosphotyrosyl content of CD3 immunoprecipitated from parental BYDP cells to that from cells expressing HA-SHP-1 or HA-SHP-1C. After TCR/CD3 plus CD4 stimulation, we observed an increase in the tyrosyl phosphorylation of CD3 immunoprecipitated from parental BYDP and cells expressing HA-SHP-1C. By contrast, tyrosyl phosphorylation of CD3 immunoprecipitated from cells expressing HA-SHP-1 cells never increased above the basal level (Fig. 7B). These data indicate that SHP-1 can negatively regulate one of the very earliest TCR signaling events, phosphorylation of the CD3-chains, and that adequate localization of SHP-1 to the lipid rafts is necessary for this effect.
Expression of SHP-1and SHP-1RK30/136, but not SHP-1C, inhibits IL-2 production in response to TCR stimulation
We next examined IL-2 production in response to TCR stimulation in BYDP-HA-SHP-1 and BYDP-HA-SHP-1RK30/136 cells. Parental BYDP cells responded to stimulation via varying concentrations of plate-bound anti-CD3 by producing IL-2 in a dose-dependent manner (Fig. 8), whereas stable expression of either HA-SHP-1 or HA-SHP-1RK30/136 in BYDP cells resulted in a complete loss of IL-2 production after anti-CD3 stimulation (Fig. 8). Conversely, cells expressing HA-SHP-1C produced IL-2 comparably to parental BYDP in response to anti-CD3 stimulation (Fig. 8), indicating that impaired localization of HA-SHP-1C to lipid rafts correlates with a lack of an effect on TCR signaling events leading to IL-2 production. All cell lines produced comparable amounts of IL-2 when the requirement for early TCR-mediated signaling was bypassed via treatment with PMA plus ionomycin (Fig. 8). This suggested that SHP-1 only affected proximal TCR-mediated signaling events and that there was no aberration in the ability of the SHP-1-expressing cells to produce IL-2. Taken together, the failure of HA-SHP-1C expression to elicit the effects observed with HA-SHP-1 or HA-SHP-1RK30/136 expression indicates that localization of SHP-1 to the lipid rafts via its C terminus is functionally critical for SHP-1 to act as a negative regulator of early events in signaling through the TCR.
FIGURE 8. Expression of HA-SHP-1 or HA-SHP-1RK30/136, but not HA-SHP-1C, inhibits IL-2 production. Top panel, Parental or HA-tagged SHP-1 variants expressing BYDP cells (5 x 104) were stimulated with the indicated concentrations of plate-bound anti-CD3. Twenty-four hours later, supernatants were harvested and analyzed for IL-2 content by ELISA. All experiments were performed in triplicate. Data shown are the averages of several experiments using independent clones: parental BYDP cells, five independent experiments; BYDP-HA-SHP-1, three independent clones; BYDP-HA-SHP-1C, four independent clones; BYDP-HA-SHP-1RK30/136, two independent clones. Error bars represent the SD of the mean. Bottom panel, Standard curve generated from indicated concentrations of rIL-2 (n = 15). Error bars represent the SD of the mean.
Discussion
Although it has been well established that the protein tyrosine phosphatase SHP-1 serves as a negative regulator of TCR-mediated signaling (reviewed in Refs. 45 , 46 , and 48), how this relates to lipid raft-dependent activation events seen downstream of the TCR has not been understood. In this study we report that a fraction of endogenous SHP-1 localizes basally to the lipid rafts before stimulation through TCR/CD3 plus CD4. The basal/constitutive localization of SHP-1 in the lipid rafts and the lack of a detectable change in its levels postactivation suggest that SHP-1 regulation of TCR-mediated signaling might depend on a qualitative, rather than quantitative, change in SHP-1. However, our data do not rule out the possibility that SHP-1 might shuttle between the cytosol and the lipid rafts, but argue against a directional movement of SHP-1 in response to TCR-mediated stimulation. The constant presence of SHP-1 in the lipid rafts suggests that its activity is controlled by a mechanism other than, or perhaps in addition to, its subcellular localization.
SHP-1 lacks any of the known sequences for post-translational modifications, such as acylation or palmitoylation, that have been described for other lipid raft-targeted proteins (69). Therefore, either a novel modification or a protein-protein or protein-lipid interaction most likely mediates the localization of SHP-1 to lipid rafts. It has been reported that SHP-1 can bind via its SH2 domains and its C terminus to phospholipids, such as phosphatidic acid, and can be activated by this binding in vitro (70). However, although phosphatidylinositol 4,5-bisphosphate is enriched in detergent-resistant membrane fractions (71), phospholipids are, in general, thought to locate to the more fluid phase of the membrane surrounding the lipid rafts. Thus, whether lipids play a role in targeting of SHP-1 remains to be seen. Alternatively, SHP-1 might localize to the lipid rafts via a protein-protein interaction. For example, it has been reported that in human T cells, SHP-1 stably associates with the leukocyte-associated Ig-like receptor 1 (72, 73). However, no such association has been found between SHP-1 and the recently identified murine homologue of leukocyte-associated Ig-like receptor 1 (74). It is also possible that SHP-1 carries an as yet uncharacterized post-translational modification that facilitates lipid rafts targeting (see below). Although a large body of work supports a key role for lipid rafts in productive signaling via the TCR, additional experimentation and perhaps newer techniques may be needed to resolve some of the controversies in the literature. These controversies were comprehensively discussed in a recent review by Munro (75), in which it was emphasized that the existence and function of lipid rafts in initiating and propagating signal transduction events downstream of receptors are less established than often assumed and that they are to some extent still speculative. Although resolving these controversies will be important, it was not the focus of our study. Our work shows that based on current understanding of lipid rafts, SHP-1 can play a role in regulating TCR signaling via its localization to the lipid rafts.
Our observation that the C-terminal residues of SHP-1 are important for its localization to the lipid rafts raises several interesting points. Activation of SHP-1 has been proposed to occur via release of an intramolecular association between the N-terminal SH2 domain and the phosphatase domain (76, 77, 78). Release of this inhibitory intramolecular association could be mediated by engagement of the SH2 domains of SHP-1 with phosphoproteins within the lipid rafts. Additionally, a recent report that phosphorylation of the C-terminal tyrosines of SHP-1 can serve to relieve an inhibitory intramolecular association (79) raises the possibility that a post-translational modification in the C terminus could similarly serve to relieve the inhibitory intramolecular association. Such a post-translational modification within SHP-1 is also suggested by the differential Ab recognition of lipid raft-associated vs detergent-soluble SHP-1 by an mAb raised against its C terminus (Fig. 4B). A C-terminal modification and/or changes in the phosphotyrosyl content of SHP-1 could also play a role in regulating catalytic activity by creating or disrupting new binding sites for proteins that facilitate release of the inhibitory intramolecular interaction.
It was previously reported that in Jurkat cells, SHP-1 localizes basally and during the first 20–30 min of anti-CD3 stimulation exclusively to the detergent-soluble fractions (58, 59). Although seemingly in contradiction to our results reported here, this discrepancy is attributable to the anti-SHP-1 mAb used in these studies for the detection of lipid raft-associated SHP-1. As we have shown here, the clone 52 fails to detect lipid raft-associated SHP-1 (Fig. 4B). In addition to data presented herein using the BYDP cell line and primary thymocytes, we obtained the same results from Jurkat cells and another T cell line, SL-12.12 (80) (V. C. J. Fawcett and U. Lorenz, unpublished observations). It is noteworthy that we also performed our SHP-1 localization studies in varying detergent conditions, and we observed basal localization of SHP-1 to lipid rafts in 1% Triton X-100 as well as in lower detergent concentrations (V. C. J. Fawcett and U. Lorenz, unpublished observation). Consistent with our observations, both studies show a negative regulatory effect of SHP-1 when artificially brought into the lipid rafts via fusion to a lipid raft-targeting sequence. Interestingly, although one study showed that overexpression of a fusion between full-length SHP-1 and an Lck-targeting sequence caused abrogation of TCR/CD3-mediated CD3 phosphorylation, NFAT activation, and IL-2 production (58), the other study reported that overexpression of a fusion between an SHP-1 deletion mutant lacking the SH2 domains and the LAT-targeting sequence caused a decrease in LAT phosphorylation and downstream signaling events, but did not affect Lck, ZAP70, or CD3 phosphorylation (59). It is unclear whether the discrepancies between these two studies are due to the different targeting sequences or whether deletion of the SH2 domains in the LAT fusion protein renders it partially nonfunctional. In this study we show that full-length SHP-1 localizes to lipid rafts without the addition of a lipid raft-targeting sequence and that it interferes with early TCR/CD3-mediated signaling affecting CD3 phosphorylation, ZAP70 association with the CD3-chain, LAT phosphorylation, and IL-2 production. These data support the hypothesis that SHP-1 plays a role in lipid rafts with respect to propagation and/or maintenance of signaling events initiated by engagement of the TCR/CD3 complex.
There are several possible explanations for the observed hypophosphorylation of SHP-1 within the lipid rafts. One potential explanation is that SHP-1 becomes tyrosyl phosphorylated only outside the lipid rafts. However, this seems unlikely because our previous data suggested that Lck is the primary kinase phosphorylating SHP-1 (63), and it has been reported that Lck rapidly translocates into the lipid rafts after stimulation (65). Additionally, we report here that lipid raft-localized SHP-1 becomes phosphorylated in cells treated with the phosphatase inhibitor, sodium pervanadate, and that this phosphorylation is also enhanced by prior TCR/CD3 plus CD4 cross-linking. A second possibility is that SHP-1 becomes rapidly dephosphorylated within the lipid rafts, either by autodephosphorylation or by the action of another phosphatase. We have previously reported that wild-type SHP-1 is very potent in dephosphorylating its own phosphotyrosine in vitro (63). The lack of detectable tyrosyl phosphorylation of SHP-1 in the lipid rafts during T cell stimulation could therefore potentially reflect an increased SHP-1 phosphatase activity within the lipid rafts. A third possibility is that SHP-1 becomes phosphorylated in the lipid rafts and that its phosphorylation provides a signal to become excluded from the lipid rafts with the concurrent influx of unphosphorylated SHP-1. Although formally possible, this seems less likely, because we did not detect any appreciable loss of SHP-1 protein from the lipid rafts fractions after pervanadate treatment, which resulted in SHP-1 becoming highly phosphorylated. Thus, our data suggest rapid dephosphorylation to be the most likely explanation for the observed hypophosphorylation of SHP-1 within the lipid rafts. Whether this dephosphorylation of SHP-1 within the lipid rafts is mediated by autodephosphorylation or by the action of another phosphatase will require additional studies.
The fact that in response to TCR/CD3 cross-linking, phosphorylated SHP-1 can only be found outside the lipid rafts is also interesting with respect to a recent report by Germain’s group (81). Stefanova et al. (81) showed that in response to antagonist binding, phosphorylated SHP-1 is recruited to the TCR complex where phospho-SHP-1 is bound by Lck, followed by the inactivation of Lck. It will be interesting to test whether this complex forms outside the lipid rafts or whether antagonist binding constitutes a condition where SHP-1 is phosphorylated in the lipid rafts. It is conceivable that the phosphatase that dephosphorylates SHP-1 in the lipid rafts upon TCR/CD3 engagement might not be active upon antagonist stimulation, thereby allowing SHP-1 to become phosphorylated in the lipid rafts, as we have seen under certain experimental conditions, such as pervanadate treatment.
At a functional level, we found that the expression of either the full-length protein (HA-SHP-1) or an SH2 domain mutant (HA-SHP-1RK30/136) resulted in inhibition of signaling events initiated through the TCR. This was evident based on a lack of CD3 chain phosphorylation, loss of ZAP70 recruitment to the TCR/CD3 complex, absence of LAT phosphorylation, and inhibition of IL-2 production in response to stimulation. Because it has been proposed that SHP-1 becomes activated via engagement of its SH2 domains with phosphoprotein, it is interesting that expression of the SH2 domain mutant, HA-SHP-1RK30/136, inhibits TCR-mediated signaling events. Although it is possible that addition of the HA tag relieves the inhibitory intramolecular association without the need of an engagement of the SH2 domains, the precise mechanism by which this mutant is functional requires additional investigations. In this context, it is notable that overexpression of HA-SHP-1 at levels comparable with or even below endogenous levels caused complete abrogation of early TCR signaling events. In addition, we observed that the HA-tagged, full-length SHP-1 has a much higher basal phosphatase activity than the endogenous wild-type protein when assessed in in vitro phosphatase assays (data not shown). Taken together, these data indicate that the added HA tag might relieve an intramolecular inhibitory mechanism, thereby potentially creating an activated or hyperactive SHP-1 protein.
In contrast to the inhibitory effects on TCR-mediated signaling events observed with HA-SHP-1 or HA-SHP-1RK30/136 expression in BYDP cells, the expression of HA-SHP-1C failed to elicit any such inhibitory effects. There are several potential explanations. One possibility is that the deletion of 68 aa from the C terminus renders HA-SHP-1C catalytically inactive. However, in vitro phosphatase assays show that the HA-SHP-1C does possess catalytic activity (data not shown). This is consistent with previous studies in which analogous deletions of the C-terminal residues did not negatively affect its catalytic activity (P. D. Lyons and U. Lorenz, unpublished observation) (77, 82). A more likely explanation for the lack of effect of HA-SHP-1C expression on signaling events initiated through the TCR is the protein’s insufficient localization to lipid rafts (Fig. 5B). Correlation of the null effect of HA-SHP-1C expression with its poor localization to lipid rafts indicates that the localization of SHP-1 to the lipid rafts is critical to its role in the negative regulation of TCR-mediated signaling events.
Although we previously observed that overexpression of a GST-SHP-1 protein in BYDP cells did not have a significant effect on IL-2 production (52), we observe that the expression of full-length HA-SHP-1 potently inhibits IL-2 production. However, upon further characterization of the GST-SHP-1 cell line, we discovered that although the GST-SHP-1 is expressed 3- to 5-fold greater than endogenous SHP-1, this GST-tagged protein is poorly localized to the lipid rafts (V. C. J. Fawcett and U. Lorenz, unpublished observation). This effect is most likely attributable to some aspect of the physical structure of the GST tag hindering localization to the tightly ordered lipid rafts, because HA-tagged, full-length SHP-1 localized to the lipid rafts comparably to endogenous SHP-1 (Fig. 5B, top left panels). The poor localization of the GST-tagged SHP-1 and its deficiency in inhibition of TCR-mediated signaling events, which is analogous to the results seen with the HA-SHP-1C mutant, underscores lipid raft localization as being essential to the ability of SHP-1 to function as a negative regulator of TCR-mediated signaling. This concept is also supported by prior reports in which targeting SHP-1 to lipid rafts resulted in the disruption of TCR-mediated signaling events (58, 59).
Our data imply that the negative regulatory role of SHP-1 on signaling through the TCR/CD3 complex is mediated within the lipid rafts. Although the substrates of SHP-1 in T cells remain unclear, our data suggest that SHP-1 acts to regulate proteins involved in early TCR-mediated signaling events, such as Lck and/or the CD3 chain. Although the CD3 chain remains a potential SHP-1 substrate, recent data implicate another phosphatase, PTPH1, in CD3 chain dephosphorylation (57).
In this study we attempted to elucidate whether there are physical and/or functional differences in SHP-1 when localized to the lipid rafts that may contribute to the role of SHP-1 as a negative regulator of signaling through the TCR. Our data provide evidence that there is a physical difference in the C terminus of SHP-1 when localized to the lipid rafts. Moreover, our results indicate that the C terminus of SHP-1 is important in mediating the localization of SHP-1 to the lipid rafts, and that it is thereby critical for the inhibitory role of SHP-1 on signaling events initiated through the TCR. This also implies an indirect, previously unidentified, role for the C terminus of SHP-1.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We are grateful to members of the laboratory and to Dr. Kodi Ravichandran for critical reading of the manuscript and their comments and suggestions.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grant RO1AI48672.
2 Address correspondence and reprint requests to Dr. Ulrike Lorenz, Department of Microbiology, Jordan Hall 7212, University of Virginia Health System, P.O. Box 800734, Charlottesville, VA 22908-0734. E-mail address: ulorenz{at}virginia.edu
3 Abbreviations used in this paper: SH2, Src homology 2; HA, hemagglutinin; LAT, linker for activation of T cell; SHP-1, SH2 domain-containing phosphatase 1.
Received for publication August 13, 2004. Accepted for publication December 17, 2004.
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The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) has previously been shown to be a negative regulator of signaling mediated via the TCR. A growing body of evidence indicates that the regulated localization of proteins within certain membrane subdomains, referred to as lipid rafts, is important for the successful transduction of signaling events downstream of the TCR. However, considerably less is known about the localization of negative regulators during these lipid raft-dependent signaling events. In this study we have investigated the subcellular localization of SHP-1 and its role in regulation of TCR-mediated signaling. Our studies demonstrate that in a murine T cell hybridoma as well as in primary murine thymocytes, a fraction of SHP-1 localizes to the lipid rafts, both basally and after TCR stimulation. Interestingly, although SHP-1 localized in the nonraft fractions is tyrosine phosphorylated, the SHP-1 isolated from the lipid rafts lacks the TCR-induced tyrosine phosphorylation, suggesting physical and/or functional differences between these two subpopulations. We identify a requirement for the C-terminal residues of SHP-1 in optimal localization to the lipid rafts. Although expression of SHP-1 that localizes to lipid rafts potently inhibits TCR-mediated early signaling events and IL-2 production, the expression of lipid raft-excluded SHP-1 mutants fails to elicit any of the inhibitory effects. Taken together these studies reveal a key role for lipid raft localization of SHP-1 in mediating the inhibitory effects on T cell signaling events.
Introduction
Signaling through the TCR is an event that is critical for both T cell development and mature T cell function. Activation of T cells through the TCR induces tyrosine phosphorylation of numerous intracellular proteins and evokes a signaling cascade that, depending on the differentiation state of the T lymphocyte, promotes proliferation, differentiation, or cell death. One of the initial events that occurs after stimulation through the TCR is the rapid tyrosine phosphorylation of ITAM motifs (ITAMs) residing in the cytoplasmic domains of the CD3 chains; this phosphorylation is mediated by the Src family protein tyrosine kinases Lck and Fyn (reviewed in Refs. 1, 2, 3). Simultaneous engagement of the coreceptor CD4 or CD8 amplifies the signal by increasing the amount of activated Lck in close proximity to the TCR (reviewed in Refs. 1 and 3). ZAP70, a Syk/ZAP70 family nontransmembrane protein tyrosine kinase, binds via its Src homology 2 (SH2)3 domains to the phosphorylated ITAMs of CD3 and is subsequently activated by Lck and/or Fyn (reviewed in Refs. 2 and 3). ZAP70 then transmits and amplifies downstream signals by facilitating the tyrosine phosphorylation of proteins such as linker for activation of T cells (LAT), phospholipase C1, Vav, and SH2 domain-containing leukocyte protein of 76 kDa (reviewed in Refs. 2, 3, 4, 5, 6, 7).
During T cell signaling, a critical role has been recognized for one type of membrane subdomain, often referred to as lipid rafts (reviewed in Refs. 8, 9, 10, 11). Enrichment in cholesterol and glycosphingolipids allows these microdomains a tightly packed, highly ordered structure within the more fluid plasma membrane (reviewed in Refs. 12, 13, 14). Lipid rafts have been defined biochemically as being insoluble in nonionic detergent at 4°C (reviewed in Refs. 15, 16, 17). In addition to the biochemical studies, various microscopic techniques have allowed visualization of lipid rafts and provided further evidence that some molecules are constitutively anchored in lipid rafts, whereas others undergo a regulated traffic to the lipid rafts (18, 19, 20, 21, 22, 23, 24, 25 ; reviewed in Refs. 11 , 26 , and 27). It has recently been shown that a number of proteins involved in signaling through the TCR localize to the lipid rafts either constitutively, such as CD4, CD8, Lck, Fyn, LAT, phosphoprotein associated with GEMS/Csk-binding protein, and LIME (20, 28, 29, 30, 31, 32, 33, 34, 35), or in a stimulus-dependent manner, such as ZAP70, Shc, SH2 domain-containing leukocyte protein of 76 kDa, Grb2-related adaptor downstream of Shc, and phospholipase C1 (29, 35, 36). Other proteins, such as Csk and the transmembrane phosphatase CD45 (30, 35, 37, 38, 39), appear to be excluded from the lipid rafts after activation. The importance of lipid rafts in T cell signaling was also emphasized by several studies illustrating that disruption of lipid rafts and/or displacement of signaling molecules from the lipid rafts abolishes TCR-mediated signaling events (29, 36, 40, 41, 42, 43, 44).
Although much is known about the initiation of TCR-mediated signaling events, relatively less is known about the mechanism(s) by which signaling is negatively controlled or terminated (reviewed in Refs. 45, 46, 47). The dephosphorylation of cellular phosphoproteins is considered to be an important part of this process, because the rapid rise in intracellular tyrosine phosphorylation that occurs upon T cell stimulation is transient, decreasing within minutes of the initial response. Given that tyrosine phosphorylation declines rapidly after the initial stimulation response, protein tyrosine phosphatases might reside or be recruited to the lipid rafts to rapidly terminate signaling. Numerous studies conducted in primary thymocytes, T cell lines, and T cell hybridomas have revealed the protein tyrosine phosphatase SH2 domain-containing phosphatase 1 (SHP-1) as a negative regulator of TCR-mediated signaling (reviewed in Ref. 45, 46 , and 48). Based on studies using T lymphocytes derived from the SHP-1-negative motheaten mice (49, 50) and cell lines overexpressing dominant negative mutants of SHP-1 (51, 52), SHP-1 does not seem to affect basal tyrosine phosphorylation and therefore probably exists basally in an inactive state. After TCR stimulation, SHP-1 is thought to become activated, potentially by engagement of its SH2 domains with an as yet unidentified partner and is then able to act on potential targets, such as Lck (49, 53), ZAP70 (51, 54), Vav (55), PI3K (56), and potentially the CD3 chains (57). Because SHP-1 has been shown to be involved in the negative regulation of signaling events initiated through the TCR, and many of its proposed substrates localize to the lipid rafts, we investigated whether SHP-1 localizes to lipid rafts, and how such localization may be functionally important. Interestingly, in two recent studies, SHP-1 appeared to not localize by itself to the lipid rafts. However, targeting SHP-1 to the lipid rafts inhibited TCR-mediated signaling events, indicating that a role for SHP-1 may indeed exist within the lipid rafts (58, 59).
In this study we demonstrate that in a murine T cell hybridoma and in primary murine thymocytes, a fraction of SHP-1 localized to the lipid rafts. Several lines of investigation suggest that localization of SHP-1 to the lipid rafts is mediated via its C terminus and is essential for the negative regulatory role of SHP-1 in TCR-mediated signaling.
Materials and Methods
Cell culture, generation of cell lines, and primary thymocytes
Parental BYDP (60) and BYDP transfectants stably expressing hemagglutinin (HA)-tagged SHP-1 were grown in RPMI 1640 medium supplemented with 10% FBS, 5 x 10–5 M 2-ME, 2 mM L-glutamine, 10 U/ml penicillin, and 10 μg/ml streptomycin (complete medium). Cell lines expressing HA-tagged SHP-1 variants were maintained under the selection of 1 mg/ml G418.
Stable cells lines were generated by cotransfection of parental BYDP cells with pEBB vectors (61) encoding HA-tagged SHP-1 full-length or mutants and pMH-neo (62), followed by selection for G418 resistance (2 mg/ml). Mutants of SHP-1 were generated via PCR-based mutagenesis. The C terminus of SHP-1 was deleted by replacing the codon for the amino acid at position 527 with a stop codon. The phosphoprotein binding capability of the SHP-1 SH2 domains was disrupted by replacing the codons for conserved arginine residues at positions 30 and 136 with codons for lysine. Two to nine independent clones were isolated for each construct based on expression of the fusion protein.
Murine thymi were obtained from 4- to 8-wk-old C3HeB/FeJLe mice, and single cell suspensions were prepared as described previously (49). Isolated thymocytes were rested for 7 h at 37°C in complete medium (2 x 106 cells/ml) before stimulation.
T cell stimulation and lipid raft isolation
BYDP cells or primary murine thymocytes (6.5 x 107/time point) were used and stimulated as described previously (63). Briefly, cells were costimulated through TCR/CD3 plus CD4 using 1 μg/ml 145-2c11 (Southern Biotechnology Associates) in combination with either 0.5 μg/ml Leu3a anti-human CD4 (BD Biosciences) for BYDP or 1 μg/ml L3T4 anti-mouse CD4 (BD Pharmingen and Southern Biotechnology Associates) for murine thymocytes, followed by secondary Ab cross-linking via the addition of 10 μg/ml goat anti-mouse Ig (Southern Biotechnology Associates). After incubation at 37°C for the indicated times, cells were washed twice in cold PBS containing 1 mM Na3VO4, and lysed at 4°C in 1 ml of TNE lysis buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 5 mM EDTA) containing 0.5% Triton X-100, phosphatase inhibitors (5 mM NaF, 1 mM Na3VO4, and 30 mM -glycerophosphate), and protease inhibitors (10 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml antipain, and 20 μg/ml PMSF). Cell lysates were mixed with an equal volume of 80% sucrose solution. The resultant 40% sucrose solutions containing the lysates were overlaid with 2 ml of 30% sucrose solution and 1 ml of 5% sucrose solution. All sucrose solutions were made up in TNE buffer, maintained at 4°C, and freshly supplemented with phosphatase inhibitors. After ultracentrifugation in an SW55Ti rotor at 200,000 x g for 18 h at 4°C, 11 equal fractions were harvested top to bottom from each gradient. Aliquots of each fraction harvested from sucrose gradients were analyzed by 8% SDS-PAGE and subsequent immunoblotting on Immobilon-P polyvinylidene difluoride membrane (Millipore). Based on the presence of the lipid rafts marker LAT (28) and the lipid rafts-excluded transmembrane protein CD45 (35, 37, 38), fractions 2–4 and 9–10 were identified and subsequently combined as lipid rafts and detergent-soluble fractions, respectively. All pairs of lipid rafts and detergent-soluble fractions presented in the figures were run on the same gel and exposed for the same length of time.
Immunoprecipitation and immunoblotting
Equal aliquots of the combined lipid rafts fractions and detergent-soluble fractions were used for additional analyses (3.75% for analyses of whole fractions and 37.5% for immunoprecipitations). For TCR/CD3 complex precipitations from unfractionated lysates, 107 cells were stimulated by Ab cross-linking, followed by lysis in TNE buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 5 mM EDTA) including protease and phosphatase inhibitors and containing 0.5% Triton X-100 for ZAP70 coprecipitation or 1% Nonidet P-40 for CD3-chain precipitation. Lysates were cleared of nuclear debris by ultracentrifugation at 100,000 x g in a TLA-45 rotor for 20 min at 4°C. Immunoprecipitations were performed as described previously (52, 63) using polyclonal rabbit anti-SHP-1 Abs, monoclonal mouse anti-CD3 IgG1 (Santa Cruz Biotechnology), or polyclonal rabbit anti-LAT Abs (Upstate Biotechnology). Each immunoprecipitation was performed using 2 μg of Abs, because this was the amount determined to effectively clear the lysates. Immune complexes were collected for 1 h at 4°C by addition of 50 μl of protein A-Sepharose beads (Amersham Biosciences) for anti-SHP-1 and anti-LAT immunoprecipitations, or 50 μl of protein G-Sepharose beads (Amersham Biosciences) precoated with goat anti-mouse Abs (Southern Biotechnology Associates) for anti-CD3 immunoprecipitations, followed by five washes with lysis buffer containing phosphatase inhibitors. Immunoprecipitates were resolved by SDS-PAGE and subsequent immunoblotting on Immobilon-P polyvinylidene difluoride membrane (Millipore).
Immunoblottings were performed as described previously (52, 63) using the following primary Abs: mouse anti-phosphotyrosine mAb 4G10 (0.5 μg/ml), polyclonal rabbit anti-LAT Abs (1 μg/ml; Upstate Biotechnology), mouse anti-CD45 mAb (0.5 μg/ml), mouse anti-Lck mAb (1/500), mouse anti-ZAP70 mAb (0.5 μg/ml; BD Transduction Laboratories), and mouse anti-CD3 mAb (0.2 μg/ml; Santa Cruz Biotechnology). SHP-1 immunoblottings were performed using mouse anti-SHP-1 mAb clone 1SH01 (1.33 μg/ml; Neomarkers), with the following exceptions: anti-SHP-1 mAb clone 52 (0.5 μg/ml; BD Transduction Laboratories) and polyclonal rabbit anti-SHP-1 Abs (1 μg/ml; Upstate Biotechnology) were used for comparative SHP-1 Ab detection studies, as indicated in the text. Immunoblots were incubated with the appropriate HRP-conjugated secondary Abs (anti-mouse or anti-rabbit, 1/7500 dilution; Amersham Biosciences) before visualization using ECL.
Pervanadate treatment
A 40 mM stock solution of pervanadate was freshly prepared for each experiment. Equimolar amounts of Na3VO4 and H2O2 were combined and incubated for 20 min at room temperature, followed by addition of an equal volume of 100 mM Tris (pH 7.6), 100 mM HEPES (pH 7.4) containing 4 μg of catalase, and incubation for 30 min at room temperature. Pervanadate treatment was performed by incubating BYDP cells for 5 min at room temperature with a final concentration of 5 mM pervanadate. When pervanadate treatment was used in combination with stimulation via Ab cross-linking, cells were stimulated before treatment with pervanadate.
IL-2 ELISA
Cells (5 x 104) were seeded in 200 μl of complete medium/well of a flat-bottom, 96-well plate coated with the indicated concentrations of the anti-CD3 mAb, 145-2c11 (Southern Biotechnology Associates). As a positive control, PMA (5 ng/ml) and ionomycin (1 μM) were added to cells in uncoated wells. After incubation at 37°C for 24 h, 125 μl of supernatant was harvested from each well. Supernatants (50 μl) were diluted 1/1 with assay diluent (PBS/10% FBS, pH 7.0) and analyzed for IL-2 by ELISA (Mouse IL-2 OptEIA ELISA Set and TMB Substrate Reagent Set; BD Biosciences; used as described by manufacturer). Known concentrations of rIL-2 were used as standards.
Results
SHP-1 localizes to lipid rafts
To determine the basal subcellular localization of SHP-1 in T lymphocytes and whether this localization changes upon stimulation through the TCR, we made use of the BYDP cell line, a murine T cell hybridoma line that mimics various aspects of thymocyte signaling (60, 64) and has previously been used to study SHP-1 and TCR signaling (52, 63). BYDP cells were stimulated via Ab cross-linking (anti-CD3/anti-CD4), followed by isolation of the lipid rafts and detergent-soluble fractions. Overall, the protein phosphotyrosine content in both lipid rafts and detergent-soluble fractions typically peaked after 10 min of stimulation and declined by 20–30 min of stimulation (Fig. 1, top panels). This pattern of tyrosyl phosphorylation of cellular proteins was consistent with previous data (52, 60, 63) and reflected an appropriate stimulation of the cells. Additionally, as observed previously (29, 35), different sets of phosphoproteins were associated with the lipid rafts and the detergent-soluble fractions.
FIGURE 1. SHP-1 localizes to lipid rafts. BYDP cells (6.5 x 107) were stimulated by Ab cross-linking for the indicated times, lysed in 0.5% Triton X-100 lysis buffer, and fractionated via a sucrose step gradient. Combined lipid rafts and detergent-soluble fractions were separated by 8% SDS-PAGE, followed by anti-phosphotyrosine immunoblotting. The blot was stripped and reprobed for the presence of SHP-1, LAT, Lck, and CD45.
Analysis for SHP-1 revealed that a fraction of SHP-1 basally localized to the lipid rafts of BYDP cells (Fig. 1). Band densitometry of a number of immunoblots revealed that 20–30% of total cellular SHP-1 was present in the lipid rafts. However, due to the difficulty in accurately quantitating the protein amounts in lipid rafts and nonlipid rafts, we will from hereon only state that a fraction of SHP-1 localized to lipid rafts. Although the amount of SHP-1 localizing to the lipid rafts was increased in some experiments after anti-CD3/anti-CD4 stimulation, this was not a consistent observation. The purity of the lipid rafts fractions was evaluated by blotting for proteins known to reside in or be excluded from the lipid rafts. Consistent with previous data, 50% of total cellular LAT (28) and 50% of total cellular Lck (31, 32) were localized to the lipid rafts. In contrast, CD45 was absent from the lipid rafts fractions, as previously reported (35, 37, 38), whereas it was readily detectable in the detergent-soluble fractions (Fig. 1).
SHP-1 is hypotyrosyl phosphorylated in lipid rafts
We have previously shown that SHP-1 becomes tyrosyl phosphorylated within its C terminus upon stimulation through TCR/CD3 plus CD4 (63). Mapping of the phosphorylation sites as well as analyses of primary thymocytes and several T cell lines have indicated that this phosphorylation is Lck dependent (63). We examined the tyrosyl phosphorylation status of SHP-1 in the lipid rafts and/or detergent-soluble fractions. Interestingly, SHP-1 was not phosphorylated on tyrosine when immunoprecipitated from the lipid rafts, but was phosphorylated in the detergent-soluble fractions of BYDP cells stimulated via TCR/CD3 plus CD4 (Fig. 2A). Because activated Lck localizes to the lipid rafts (65), and our previous data suggested that Lck is the major tyrosine kinase phosphorylating SHP-1 upon TCR/CD3 plus CD4 stimulation (63), the lack of phosphorylation on lipid raft-associated SHP-1 was surprising. To test whether this reflects a general phenomenon, the experiment was repeated in primary cells. Consistent with our observation in BYDP cells, fractionation of murine thymocytes revealed a hypophosphorylation of lipid raft-associated SHP-1 (Fig. 2B). The observed lack of tyrosyl phosphorylation of SHP-1 cannot be attributed to a general deficiency in protein tyrosyl phosphorylation in the lipid rafts, because a number of phosphoproteins are detectable in the lipid raft fractions upon TCR/CD3 plus CD4 stimulation (Fig. 1, top left panel). Hypophosphorylation of SHP-1 isolated from the lipid raft fractions compared with SHP-1 isolated from the detergent-soluble fractions suggests that there may be functional and/or physical differences between these two subpopulations of SHP-1.
FIGURE 2. SHP-1 is hypo-tyrosyl phosphorylated in lipid rafts of BYDP cells and primary murine thymocytes. BYDP cells (6.5 x 107) (A) or primary murine thymocytes (B) were stimulated by Ab cross-linking for the indicated times. 0.5% Triton X-100 cell lysates were fractionated via a sucrose step gradient. SHP-1 was immunoprecipitated from combined lipid rafts and detergent-soluble fractions, followed by 8% SDS-PAGE and immunoblotting for phosphotyrosyl content. The immunoblots were stripped and reprobed for SHP-1. *, The 70-kDa phosphoprotein migrating above SHP-1 in the detergent-soluble fractions is ZAP70 and is due to the presence of the Abs used for stimulation, rather than a direct interaction with SHP-1 (V. C. J. Fawcett and U. Lorenz, unpublished observation; refer to Results for further explanation).
The 70-kDa phosphoprotein band migrating above SHP-1 in the detergent-soluble fractions isolated from BYDP cells (Fig. 2, top right panel) was identified by immunoblotting as ZAP70 (data not shown). Additional studies suggested that the coprecipitation of ZAP70 is not due to a direct interaction with SHP-1, but results from an indirect interaction via the stimulating Abs to the TCR/CD3 complex (V. C. J. Fawcett and U. Lorenz, unpublished observation). Consistent with published reports, ZAP70 was primarily detected in the detergent-soluble fractions, where the majority of the TCR complexes are found after stimulation via TCR/CD3 Ab cross-linking (66).
Lipid raft-localized SHP-1 can be tyrosyl phosphorylated
One possible explanation for the hypophosphorylation of lipid raft-localized SHP-1 is that SHP-1 cannot be tyrosyl phosphorylated within the lipid rafts. This could be due to a lack of kinases within the lipid rafts or to SHP-1 not being accessible to the available kinases. Moreover, it would imply that there is very limited movement of SHP-1 into or out of the lipid rafts. To determine whether tyrosine phosphorylation of SHP-1 can be achieved at all within the lipid raft environment, we used the tyrosine phosphatase inhibitor pervanadate to achieve maximal, albeit unregulated, tyrosine phosphorylation (67). Treatment of BYDP cells with pervanadate resulted in a rapid and robust tyrosine phosphorylation of cellular proteins (Fig. 3A). An even greater amount of tyrosine phosphorylation was achieved by stimulating the cells via Ab cross-linking (anti-CD3/anti-CD4) before pervanadate treatment, indicating that the ideal localization of either the kinases or the substrates is enhanced by and/or dependent on TCR stimulation. Treatment of BYDP cells with pervanadate resulted in an intense tyrosine phosphorylation of SHP-1 in both the lipid rafts and detergent-soluble fractions (Fig. 3B). Stimulating the cells through TCR/CD3 plus CD4 before pervanadate treatment further augmented the tyrosine phosphorylation of SHP-1 in both the lipid rafts and detergent-soluble fractions (Fig. 3B). Based on these data, we conclude that the C terminus of SHP-1 is accessible to and capable of being tyrosyl phosphorylated within the lipid rafts. Thus, the hypophosphorylation of SHP-1 within the lipid rafts compared with the detergent-soluble fractions is most likely attributable to a heightened dephosphorylation of SHP-1 within the lipid rafts, either via autodephosphorylation or through the action of another phosphatase.
FIGURE 3. SHP-1 is tyrosyl phosphorylated in the lipid rafts after treatment of cells with pervanadate. BYDP cells (6.5 x 107) were treated with 5 mM pervanadate alone or after stimulation by Ab cross-linking. Triton X-100 (0.5%) cell lysates were fractionated via a sucrose step gradient. A, Combined lipid rafts and detergent-soluble fractions were separated by 8% SDS-PAGE and subjected to anti-phosphotyrosine immunoblotting. B, SHP-1 was immunoprecipitated from combined lipid rafts and detergent-soluble fractions, followed by 8% SDS-PAGE and immunoblotting for phosphotyrosyl content. The immunoblots were stripped and reprobed for SHP-1. *, The 70-kDa phosphoprotein migrating above SHP-1 is ZAP70.
Differential Ab detection of lipid rafts-localized SHP-1
During the course of our studies, we made an interesting discovery regarding the C-terminal domain of SHP-1. Compared with anti-SHP-1 polyclonal Abs, an mAb clone 52 (directed against the C terminus of SHP-1; commercially available from BD Transduction Laboratories) displayed a diminished recognition of the highly phosphorylated form of SHP-1 that exists after pervanadate treatment (Fig. 4A, compare middle and bottom panels, lane 4). Additionally, we found that this mAb failed to recognize SHP-1 isolated from the lipid rafts, whereas it readily detected SHP-1 isolated from the detergent-soluble fractions (Fig. 4B, top panels). We were able to detect SHP-1 in the lipid rafts fractions using polyclonal anti-SHP-1 Abs (Fig. 4B, bottom left panel). Notably, there was no difference between the monoclonal and the polyclonal anti-SHP-1 Abs in detecting not hyperphosphorylated SHP-1 isolated from the detergent-soluble fractions (Fig. 4A, lanes 1–3, and Fig. 4B, right panels), indicating that the discrepancy between these Abs is not simply due to a decreased sensitivity of mAb clone 52. This mAb, which recognizes a C-terminal epitope in SHP-1, fails to detect highly phosphorylated SHP-1, most likely because the epitope is masked by the tyrosyl phosphorylation in the C terminus. Because the lipid raft-associated SHP-1 is hypophosphorylated, we hypothesized that this subpopulation of SHP-1 may possess a different posttranslational modification in the C-terminal region, which interferes with recognition by the mAb, and that such a modification could be involved in the localization of SHP-1 to the lipid rafts and/or its function within the lipid rafts.
FIGURE 4. An mAb raised against the C terminus of SHP-1 fails to recognize SHP-1 when either hyperphosphorylated or lipid raft-localized. A, BYDP cells (6.5 x 107) were stimulated by Ab cross-linking and/or 5 mM pervanadate treatment as indicated. Triton X-100 (0.5%) cell lysates were fractionated via a sucrose step gradient, and SHP-1 was immunoprecipitated from the combined detergent-soluble fractions, followed by 8% SDS-PAGE and immunoblotting for phosphotyrosine. *, ZAP70. The immunoblot was stripped and reprobed for SHP-1 using an mAb (clone 52) directed against the C terminus of SHP-1. The immunoblot was stripped again and reprobed for SHP-1 using rabbit polyclonal Abs against SHP-1. B, BYDP cells (6.5 x 107) were stimulated by Ab cross-linking for the indicated times, lysed in 0.5% Triton X-100 lysis buffer, and fractionated via a sucrose step gradient. SHP-1 was immunoprecipitated from the combined lipid rafts and detergent-soluble fractions. Immunoprecipitates were resolved by 8% SDS-PAGE and subjected to immunoblotting for SHP-1 using an mAb (clone 52) directed against the C terminus of SHP-1. The immunoblot was stripped and reprobed for SHP-1 using rabbit polyclonal Abs against SHP-1.
Subcellular localization of mutants of SHP-1
Unlike other lipid raft-localized proteins, such as LAT and Lck, SHP-1 contains no known lipid raft localization sequences. We therefore asked which region/domain(s) of SHP-1 was essential for its localization to lipid rafts by analyzing the subcellular localization of mutants of SHP-1 in BYDP cells. Based on the above observations, we considered a role for the C terminus of SHP-1 in mediating localization to lipid rafts. We generated N-terminally HA-tagged versions of full-length SHP-1 (denoted HA-SHP-1) and a C-terminal deletion mutant (denoted HA-SHP-1C), which lacks the last 68 aa of SHP-1, including the C-terminal tyrosine phosphorylation sites, but does not affect the catalytic domain (see schematic in Fig. 5A).
FIGURE 5. Deletion of the C terminus of SHP-1 impairs localization to the lipid rafts. A, Schematic representation of the versions of HA-tagged full-length and mutant SHP-1 introduced into BYDP cells. B, BYDP cells (6.5 x 107) stably expressing the indicated HA-tagged SHP-1 variants were stimulated by Ab cross-linking for the indicated times, lysed in 0.5% Triton X-100 lysis buffer, and fractionated via a sucrose step gradient. Combined lipid rafts and detergent-soluble fractions were separated by 8% SDS-PAGE and analyzed for the presence of SHP-1 by immunoblotting. Two representative independent clones are shown for each HA-tagged SHP-1 variant.
When HA-SHP-1 was stably expressed in BYDP cells, we found that the relative amounts of total cellular HA-SHP-1 that localized to the lipid rafts and detergent-soluble fractions were comparable to those of endogenous SHP-1, indicating that the HA tag itself did not affect subcellular localization of SHP-1 (Fig. 5B, top left panels). Examination of the subcellular localization of HA-SHP-1C showed that this protein poorly localized to the lipid rafts compared with endogenous SHP-1 or HA-SHP-1 (Fig. 5B, bottom left panels), suggesting that the C terminus of SHP-1 is involved in mediating localization to the lipid rafts. As a control and to test the possible involvement of the SH2 domains of SHP-1 in lipid raft localization, we also generated SHP-1 with point mutations within the SH2 domains, denoted HA-SHP-1RK30/136. The subcellular localization of HA-SHP-1RK30/136 was comparable to that of endogenous SHP-1 (Fig. 5B, top right panels), indicating that the localization of SHP-1 to the lipid rafts is not dependent on functional SH2 domains. However, as observed for the HA-SHP-1C mutant, deletion of the C-terminal 68 aa from the SH2 mutant (denoted HA-SHP-1RK30/136/C) impaired its ability to localize to lipid rafts (Fig. 5B, bottom right panels). These findings implicate a role for the C terminus in mediating the localization of SHP-1 to the lipid rafts and raise the possibility that the C terminus of SHP-1 might possess a novel lipid raft-targeting modification.
Expression of SHP-1and SHP-1RK30/136, but not SHP-1C, affects signaling events initiated through the TCR
We next evaluated the functional consequences of expressing SHP-1 mutants in BYDP cells. It has been previously reported that targeting SHP-1 to the lipid rafts inhibits TCR-mediated signaling events (58, 59). Because the expression of HA-SHP-1 or HA-SHP-1RK30/136 in BYDP cells results in increased representation of SHP-1 within the lipid rafts without artificial targeting to the lipid rafts (Fig. 5B), we asked whether functional consequences would ensue.
We first compared the phosphoprotein profiles of lipid raft-associated and detergent-soluble proteins in the lysates of parental BYDP cells to those expressing HA-SHP-1, HA-SHP-1C, or HA-SHP-1RK30/136 upon TCR/CD3 plus CD4 stimulation. Interestingly, the expression of the poorly lipid raft-localized HA-SHP-1C mutant had no effect on overall tyrosine phosphorylation, whereas expression of HA-SHP-1 and HA-SHP-1RK30/136 resulted in several alterations in the overall phosphoprotein patterns in both the lipid rafts and the detergent-soluble fractions (Fig. 6A, as indicated by arrows). One of the most affected phosphoproteins was in the 35- to 40-kDa range, where a prominent inducible phosphoprotein band was lost from both the lipid rafts and the detergent-soluble fractions in cells expressing HA-SHP-1 or HA-SHP-1RK30/136 (Fig. 6A).
FIGURE 6. Expression of HA-SHP-1 or HA-SHP-1RK30/136, but not HA-SHP-1C, results in the loss of LAT tyrosyl phosphorylation. Parental or HA-tagged SHP-1 variants expressing BYDP cells (6.5 x 107) were stimulated by Ab cross-linking for the indicated times, lysed in 0.5% Triton X-100 lysis buffer, and fractionated via a sucrose step gradient. A, Combined lipid rafts and detergent-soluble fractions were separated by 8% SDS-PAGE and subjected to anti-phosphotyrosine immunoblotting. B, LAT was immunoprecipitated from combined lipid rafts and detergent-soluble fractions. Immunoprecipitates were resolved by 8% SDS-PAGE, followed by immunoblotting for phosphotyrosine and LAT. The SHP-1 expression levels of these representative clones are shown in the bottom panels of Fig. 5B (labeled clone 2).
Because LAT is one of the major proteins phosphorylated in response to TCR stimulation, we hypothesized that this 35- to 40-kDa phosphoprotein band might be LAT. Although phosphorylated LAT is detectable in both the lipid rafts and detergent-soluble fractions after TCR stimulation, previous studies have determined that its phosphorylation takes place within the lipid rafts (28, 68). Therefore, a loss of LAT phosphorylation in cells expressing HA-SHP-1 and HA-SHP-1RK30/136, but not in cells expressing HA-SHP-1C in which SHP-1 is comparatively underrepresented in the lipid rafts, would suggest that SHP-1 expression elicits an inhibitory effect only when localized to lipid rafts. LAT immunoprecipitated from cells expressing HA-SHP-1 or HA-SHP-1RK30/136 displayed a complete loss of inducible tyrosine phosphorylation after TCR/CD3 plus CD4 stimulation, whereas it was readily detectable in parental BYDP (Fig. 6B). Interestingly, tyrosine phosphorylation of LAT immunoprecipitated from cells expressing SHP-1C was comparable to that observed in parental BYDP cells (Fig. 6B). This finding indicates that the inhibitory effect on LAT phosphorylation requires localization of SHP-1 to lipid rafts.
Phosphorylation of LAT requires ZAP70 recruitment to the TCR/CD3 complex. To examine whether this upstream signaling event was also affected in cells expressing HA-SHP-1 or SHP-1RK30/136, we took advantage of our prior observation that ZAP70 coprecipitates with the anti-CD3/anti-CD4 stimulating Abs from BYDP cell lysates (Figs. 2A, 3B, and 4A). Analyses of cells expressing HA-SHP-1 or HA-SHP-1RK30/136 revealed no detectable association between ZAP70 and the TCR complex (Fig. 7A). In contrast, stimulation of parental BYDP and cells expressing HA-SHP-1C readily induced association of ZAP70 with the TCR complex, an event that typically was first detectable at 5–10 min of TCR/CD3 plus CD4 stimulation, with a peak at 15–20 min of stimulation. As expected, little or no basal association of ZAP70 with the TCR/CD3 complex was observed (Fig. 7A). These results indicate that the expression of HA-SHP-1 or HA-SHP-1RK30/136 in BYDP cells inhibits the phosphorylation of LAT by interfering with ZAP70 association with the TCR complex, whereas the expression of HA-SHP-1C fails to elicit these effects, most likely due to its inefficient localization to lipid rafts.
FIGURE 7. Expression of HA-SHP-1 or HA-SHP-1RK30/136, but not HA-SHP-1C, results in a loss of ZAP70 recruitment to the TCR and loss of phosphorylation of the CD3-chains. Parental or HA-tagged SHP-1 variants expressing BYDP cells (107) were stimulated by Ab cross-linking for the indicated times and lysed in 0.5% Triton X-100 lysis buffer (A) or 1% Nonidet P-40 lysis buffer (B). A, The TCR/CD3 complex was precipitated from the cleared lysates. Precipitates were subjected to 8% SDS-PAGE and analyzed for the coprecipitation of ZAP70 by immunoblotting (top panels). Five percent of the cleared lysates were separated by 8% SDS-PAGE and analyzed for ZAP70 and SHP-1 expression (bottom panels). B, CD3 was immunoprecipitated from the cleared lysates. Immunoprecipitates were subjected to 10% SDS-PAGE and analyzed for phosphotyrosyl content by immunoblotting. Immunoblots were stripped and reprobed for CD3 (top panels). Five percent of the cleared lysates were separated by 8% SDS-PAGE and analyzed for SHP-1 expression (bottom panel).
One potential explanation for the lack of ZAP70 association with the TCR complex in cells expressing HA-SHP-1 and HA-SHP-1RK30/136 is that increased representation of SHP-1 in the lipid rafts results in a loss of phosphorylation of the CD3 ITAMs, thereby preventing recruitment of ZAP70. We therefore compared the phosphotyrosyl content of CD3 immunoprecipitated from parental BYDP cells to that from cells expressing HA-SHP-1 or HA-SHP-1C. After TCR/CD3 plus CD4 stimulation, we observed an increase in the tyrosyl phosphorylation of CD3 immunoprecipitated from parental BYDP and cells expressing HA-SHP-1C. By contrast, tyrosyl phosphorylation of CD3 immunoprecipitated from cells expressing HA-SHP-1 cells never increased above the basal level (Fig. 7B). These data indicate that SHP-1 can negatively regulate one of the very earliest TCR signaling events, phosphorylation of the CD3-chains, and that adequate localization of SHP-1 to the lipid rafts is necessary for this effect.
Expression of SHP-1and SHP-1RK30/136, but not SHP-1C, inhibits IL-2 production in response to TCR stimulation
We next examined IL-2 production in response to TCR stimulation in BYDP-HA-SHP-1 and BYDP-HA-SHP-1RK30/136 cells. Parental BYDP cells responded to stimulation via varying concentrations of plate-bound anti-CD3 by producing IL-2 in a dose-dependent manner (Fig. 8), whereas stable expression of either HA-SHP-1 or HA-SHP-1RK30/136 in BYDP cells resulted in a complete loss of IL-2 production after anti-CD3 stimulation (Fig. 8). Conversely, cells expressing HA-SHP-1C produced IL-2 comparably to parental BYDP in response to anti-CD3 stimulation (Fig. 8), indicating that impaired localization of HA-SHP-1C to lipid rafts correlates with a lack of an effect on TCR signaling events leading to IL-2 production. All cell lines produced comparable amounts of IL-2 when the requirement for early TCR-mediated signaling was bypassed via treatment with PMA plus ionomycin (Fig. 8). This suggested that SHP-1 only affected proximal TCR-mediated signaling events and that there was no aberration in the ability of the SHP-1-expressing cells to produce IL-2. Taken together, the failure of HA-SHP-1C expression to elicit the effects observed with HA-SHP-1 or HA-SHP-1RK30/136 expression indicates that localization of SHP-1 to the lipid rafts via its C terminus is functionally critical for SHP-1 to act as a negative regulator of early events in signaling through the TCR.
FIGURE 8. Expression of HA-SHP-1 or HA-SHP-1RK30/136, but not HA-SHP-1C, inhibits IL-2 production. Top panel, Parental or HA-tagged SHP-1 variants expressing BYDP cells (5 x 104) were stimulated with the indicated concentrations of plate-bound anti-CD3. Twenty-four hours later, supernatants were harvested and analyzed for IL-2 content by ELISA. All experiments were performed in triplicate. Data shown are the averages of several experiments using independent clones: parental BYDP cells, five independent experiments; BYDP-HA-SHP-1, three independent clones; BYDP-HA-SHP-1C, four independent clones; BYDP-HA-SHP-1RK30/136, two independent clones. Error bars represent the SD of the mean. Bottom panel, Standard curve generated from indicated concentrations of rIL-2 (n = 15). Error bars represent the SD of the mean.
Discussion
Although it has been well established that the protein tyrosine phosphatase SHP-1 serves as a negative regulator of TCR-mediated signaling (reviewed in Refs. 45 , 46 , and 48), how this relates to lipid raft-dependent activation events seen downstream of the TCR has not been understood. In this study we report that a fraction of endogenous SHP-1 localizes basally to the lipid rafts before stimulation through TCR/CD3 plus CD4. The basal/constitutive localization of SHP-1 in the lipid rafts and the lack of a detectable change in its levels postactivation suggest that SHP-1 regulation of TCR-mediated signaling might depend on a qualitative, rather than quantitative, change in SHP-1. However, our data do not rule out the possibility that SHP-1 might shuttle between the cytosol and the lipid rafts, but argue against a directional movement of SHP-1 in response to TCR-mediated stimulation. The constant presence of SHP-1 in the lipid rafts suggests that its activity is controlled by a mechanism other than, or perhaps in addition to, its subcellular localization.
SHP-1 lacks any of the known sequences for post-translational modifications, such as acylation or palmitoylation, that have been described for other lipid raft-targeted proteins (69). Therefore, either a novel modification or a protein-protein or protein-lipid interaction most likely mediates the localization of SHP-1 to lipid rafts. It has been reported that SHP-1 can bind via its SH2 domains and its C terminus to phospholipids, such as phosphatidic acid, and can be activated by this binding in vitro (70). However, although phosphatidylinositol 4,5-bisphosphate is enriched in detergent-resistant membrane fractions (71), phospholipids are, in general, thought to locate to the more fluid phase of the membrane surrounding the lipid rafts. Thus, whether lipids play a role in targeting of SHP-1 remains to be seen. Alternatively, SHP-1 might localize to the lipid rafts via a protein-protein interaction. For example, it has been reported that in human T cells, SHP-1 stably associates with the leukocyte-associated Ig-like receptor 1 (72, 73). However, no such association has been found between SHP-1 and the recently identified murine homologue of leukocyte-associated Ig-like receptor 1 (74). It is also possible that SHP-1 carries an as yet uncharacterized post-translational modification that facilitates lipid rafts targeting (see below). Although a large body of work supports a key role for lipid rafts in productive signaling via the TCR, additional experimentation and perhaps newer techniques may be needed to resolve some of the controversies in the literature. These controversies were comprehensively discussed in a recent review by Munro (75), in which it was emphasized that the existence and function of lipid rafts in initiating and propagating signal transduction events downstream of receptors are less established than often assumed and that they are to some extent still speculative. Although resolving these controversies will be important, it was not the focus of our study. Our work shows that based on current understanding of lipid rafts, SHP-1 can play a role in regulating TCR signaling via its localization to the lipid rafts.
Our observation that the C-terminal residues of SHP-1 are important for its localization to the lipid rafts raises several interesting points. Activation of SHP-1 has been proposed to occur via release of an intramolecular association between the N-terminal SH2 domain and the phosphatase domain (76, 77, 78). Release of this inhibitory intramolecular association could be mediated by engagement of the SH2 domains of SHP-1 with phosphoproteins within the lipid rafts. Additionally, a recent report that phosphorylation of the C-terminal tyrosines of SHP-1 can serve to relieve an inhibitory intramolecular association (79) raises the possibility that a post-translational modification in the C terminus could similarly serve to relieve the inhibitory intramolecular association. Such a post-translational modification within SHP-1 is also suggested by the differential Ab recognition of lipid raft-associated vs detergent-soluble SHP-1 by an mAb raised against its C terminus (Fig. 4B). A C-terminal modification and/or changes in the phosphotyrosyl content of SHP-1 could also play a role in regulating catalytic activity by creating or disrupting new binding sites for proteins that facilitate release of the inhibitory intramolecular interaction.
It was previously reported that in Jurkat cells, SHP-1 localizes basally and during the first 20–30 min of anti-CD3 stimulation exclusively to the detergent-soluble fractions (58, 59). Although seemingly in contradiction to our results reported here, this discrepancy is attributable to the anti-SHP-1 mAb used in these studies for the detection of lipid raft-associated SHP-1. As we have shown here, the clone 52 fails to detect lipid raft-associated SHP-1 (Fig. 4B). In addition to data presented herein using the BYDP cell line and primary thymocytes, we obtained the same results from Jurkat cells and another T cell line, SL-12.12 (80) (V. C. J. Fawcett and U. Lorenz, unpublished observations). It is noteworthy that we also performed our SHP-1 localization studies in varying detergent conditions, and we observed basal localization of SHP-1 to lipid rafts in 1% Triton X-100 as well as in lower detergent concentrations (V. C. J. Fawcett and U. Lorenz, unpublished observation). Consistent with our observations, both studies show a negative regulatory effect of SHP-1 when artificially brought into the lipid rafts via fusion to a lipid raft-targeting sequence. Interestingly, although one study showed that overexpression of a fusion between full-length SHP-1 and an Lck-targeting sequence caused abrogation of TCR/CD3-mediated CD3 phosphorylation, NFAT activation, and IL-2 production (58), the other study reported that overexpression of a fusion between an SHP-1 deletion mutant lacking the SH2 domains and the LAT-targeting sequence caused a decrease in LAT phosphorylation and downstream signaling events, but did not affect Lck, ZAP70, or CD3 phosphorylation (59). It is unclear whether the discrepancies between these two studies are due to the different targeting sequences or whether deletion of the SH2 domains in the LAT fusion protein renders it partially nonfunctional. In this study we show that full-length SHP-1 localizes to lipid rafts without the addition of a lipid raft-targeting sequence and that it interferes with early TCR/CD3-mediated signaling affecting CD3 phosphorylation, ZAP70 association with the CD3-chain, LAT phosphorylation, and IL-2 production. These data support the hypothesis that SHP-1 plays a role in lipid rafts with respect to propagation and/or maintenance of signaling events initiated by engagement of the TCR/CD3 complex.
There are several possible explanations for the observed hypophosphorylation of SHP-1 within the lipid rafts. One potential explanation is that SHP-1 becomes tyrosyl phosphorylated only outside the lipid rafts. However, this seems unlikely because our previous data suggested that Lck is the primary kinase phosphorylating SHP-1 (63), and it has been reported that Lck rapidly translocates into the lipid rafts after stimulation (65). Additionally, we report here that lipid raft-localized SHP-1 becomes phosphorylated in cells treated with the phosphatase inhibitor, sodium pervanadate, and that this phosphorylation is also enhanced by prior TCR/CD3 plus CD4 cross-linking. A second possibility is that SHP-1 becomes rapidly dephosphorylated within the lipid rafts, either by autodephosphorylation or by the action of another phosphatase. We have previously reported that wild-type SHP-1 is very potent in dephosphorylating its own phosphotyrosine in vitro (63). The lack of detectable tyrosyl phosphorylation of SHP-1 in the lipid rafts during T cell stimulation could therefore potentially reflect an increased SHP-1 phosphatase activity within the lipid rafts. A third possibility is that SHP-1 becomes phosphorylated in the lipid rafts and that its phosphorylation provides a signal to become excluded from the lipid rafts with the concurrent influx of unphosphorylated SHP-1. Although formally possible, this seems less likely, because we did not detect any appreciable loss of SHP-1 protein from the lipid rafts fractions after pervanadate treatment, which resulted in SHP-1 becoming highly phosphorylated. Thus, our data suggest rapid dephosphorylation to be the most likely explanation for the observed hypophosphorylation of SHP-1 within the lipid rafts. Whether this dephosphorylation of SHP-1 within the lipid rafts is mediated by autodephosphorylation or by the action of another phosphatase will require additional studies.
The fact that in response to TCR/CD3 cross-linking, phosphorylated SHP-1 can only be found outside the lipid rafts is also interesting with respect to a recent report by Germain’s group (81). Stefanova et al. (81) showed that in response to antagonist binding, phosphorylated SHP-1 is recruited to the TCR complex where phospho-SHP-1 is bound by Lck, followed by the inactivation of Lck. It will be interesting to test whether this complex forms outside the lipid rafts or whether antagonist binding constitutes a condition where SHP-1 is phosphorylated in the lipid rafts. It is conceivable that the phosphatase that dephosphorylates SHP-1 in the lipid rafts upon TCR/CD3 engagement might not be active upon antagonist stimulation, thereby allowing SHP-1 to become phosphorylated in the lipid rafts, as we have seen under certain experimental conditions, such as pervanadate treatment.
At a functional level, we found that the expression of either the full-length protein (HA-SHP-1) or an SH2 domain mutant (HA-SHP-1RK30/136) resulted in inhibition of signaling events initiated through the TCR. This was evident based on a lack of CD3 chain phosphorylation, loss of ZAP70 recruitment to the TCR/CD3 complex, absence of LAT phosphorylation, and inhibition of IL-2 production in response to stimulation. Because it has been proposed that SHP-1 becomes activated via engagement of its SH2 domains with phosphoprotein, it is interesting that expression of the SH2 domain mutant, HA-SHP-1RK30/136, inhibits TCR-mediated signaling events. Although it is possible that addition of the HA tag relieves the inhibitory intramolecular association without the need of an engagement of the SH2 domains, the precise mechanism by which this mutant is functional requires additional investigations. In this context, it is notable that overexpression of HA-SHP-1 at levels comparable with or even below endogenous levels caused complete abrogation of early TCR signaling events. In addition, we observed that the HA-tagged, full-length SHP-1 has a much higher basal phosphatase activity than the endogenous wild-type protein when assessed in in vitro phosphatase assays (data not shown). Taken together, these data indicate that the added HA tag might relieve an intramolecular inhibitory mechanism, thereby potentially creating an activated or hyperactive SHP-1 protein.
In contrast to the inhibitory effects on TCR-mediated signaling events observed with HA-SHP-1 or HA-SHP-1RK30/136 expression in BYDP cells, the expression of HA-SHP-1C failed to elicit any such inhibitory effects. There are several potential explanations. One possibility is that the deletion of 68 aa from the C terminus renders HA-SHP-1C catalytically inactive. However, in vitro phosphatase assays show that the HA-SHP-1C does possess catalytic activity (data not shown). This is consistent with previous studies in which analogous deletions of the C-terminal residues did not negatively affect its catalytic activity (P. D. Lyons and U. Lorenz, unpublished observation) (77, 82). A more likely explanation for the lack of effect of HA-SHP-1C expression on signaling events initiated through the TCR is the protein’s insufficient localization to lipid rafts (Fig. 5B). Correlation of the null effect of HA-SHP-1C expression with its poor localization to lipid rafts indicates that the localization of SHP-1 to the lipid rafts is critical to its role in the negative regulation of TCR-mediated signaling events.
Although we previously observed that overexpression of a GST-SHP-1 protein in BYDP cells did not have a significant effect on IL-2 production (52), we observe that the expression of full-length HA-SHP-1 potently inhibits IL-2 production. However, upon further characterization of the GST-SHP-1 cell line, we discovered that although the GST-SHP-1 is expressed 3- to 5-fold greater than endogenous SHP-1, this GST-tagged protein is poorly localized to the lipid rafts (V. C. J. Fawcett and U. Lorenz, unpublished observation). This effect is most likely attributable to some aspect of the physical structure of the GST tag hindering localization to the tightly ordered lipid rafts, because HA-tagged, full-length SHP-1 localized to the lipid rafts comparably to endogenous SHP-1 (Fig. 5B, top left panels). The poor localization of the GST-tagged SHP-1 and its deficiency in inhibition of TCR-mediated signaling events, which is analogous to the results seen with the HA-SHP-1C mutant, underscores lipid raft localization as being essential to the ability of SHP-1 to function as a negative regulator of TCR-mediated signaling. This concept is also supported by prior reports in which targeting SHP-1 to lipid rafts resulted in the disruption of TCR-mediated signaling events (58, 59).
Our data imply that the negative regulatory role of SHP-1 on signaling through the TCR/CD3 complex is mediated within the lipid rafts. Although the substrates of SHP-1 in T cells remain unclear, our data suggest that SHP-1 acts to regulate proteins involved in early TCR-mediated signaling events, such as Lck and/or the CD3 chain. Although the CD3 chain remains a potential SHP-1 substrate, recent data implicate another phosphatase, PTPH1, in CD3 chain dephosphorylation (57).
In this study we attempted to elucidate whether there are physical and/or functional differences in SHP-1 when localized to the lipid rafts that may contribute to the role of SHP-1 as a negative regulator of signaling through the TCR. Our data provide evidence that there is a physical difference in the C terminus of SHP-1 when localized to the lipid rafts. Moreover, our results indicate that the C terminus of SHP-1 is important in mediating the localization of SHP-1 to the lipid rafts, and that it is thereby critical for the inhibitory role of SHP-1 on signaling events initiated through the TCR. This also implies an indirect, previously unidentified, role for the C terminus of SHP-1.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We are grateful to members of the laboratory and to Dr. Kodi Ravichandran for critical reading of the manuscript and their comments and suggestions.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grant RO1AI48672.
2 Address correspondence and reprint requests to Dr. Ulrike Lorenz, Department of Microbiology, Jordan Hall 7212, University of Virginia Health System, P.O. Box 800734, Charlottesville, VA 22908-0734. E-mail address: ulorenz{at}virginia.edu
3 Abbreviations used in this paper: SH2, Src homology 2; HA, hemagglutinin; LAT, linker for activation of T cell; SHP-1, SH2 domain-containing phosphatase 1.
Received for publication August 13, 2004. Accepted for publication December 17, 2004.
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