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The Journal of Immunology, 2007, 179: 483-490.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Identification of a Novel Lipid Raft-Targeting Motif in Src Homology 2-Containing Phosphatase 11

Mohan Sankarshanan*, Zhong Ma{dagger}, Tessy Iype* and Ulrike Lorenz2,*,{dagger}

* Department of Microbiology and {dagger} Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The tyrosine phosphatase Src homology 2-containing phosphatase 1 (SHP-1) is a key negative regulator of TCR-mediated signaling. Previous studies have shown that in T cells a fraction of SHP-1 constitutively localizes to membrane microdomains, commonly referred to as lipid rafts. Although this localization of SHP-1 is required for its functional regulation of T cell activation events, how SHP-1 is targeted to the lipid rafts was unclear. In this study, we identify a novel, six-amino acid, lipid raft-targeting motif within the C terminus of SHP-1 based on several biochemical and functional observations. First, mutations of this motif in the context of full-length SHP-1 result in the loss of lipid raft localization of SHP-1. Second, this motif alone restores raft localization when fused to a mutant of SHP-1 (SHP-1 {Delta}C) that fails to localize to rafts. Third, a peptide encompassing the 6-mer motif directly binds to phospholipids whereas a mutation of this motif abolishes lipid binding. Fourth, whereas full-length SHP-1 potently inhibits TCR-induced tyrosine phosphorylation of specific proteins, expression of a SHP-1-carrying mutation within the 6-mer motif does not. Additionally, although SHP-1 {Delta}C was functionally inactive, the addition of the 6-mer motif restored its functionality in inhibiting TCR-induced tyrosine phosphorylation. Finally, this 6-mer mediated targeting of SHP-1 lipid rafts was essential for the function of this phosphatase in regulating IL-2 production downstream of TCR. Taken together, these data define a novel 6-mer motif within SHP-1 that is necessary and sufficient for lipid raft localization and for the function of SHP-1 as a negative regulator of TCR signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The importance of the subcellular localization of signaling molecules during TCR activation has become increasingly clear. In particular, the critical role of specific membrane microdomains for optimal TCR signaling has been recognized (reviewed in Refs. 1, 2, 3, 4). Cell membranes are composed of proteins and lipids such as cholesterol and various glycophospholipids and sphingolipids that form microdomains within the membrane. Based on their biophysical properties, glycophospholipids tend to display a mobile fluid phase, whereas sphingolipids show a more tightly packed higher organization (reviewed in Ref. 5). Moreover, gaps between the fatty acyl chains of the sphingolipids are filled with cholesterol, thereby forming a closely packed lateral lipid cluster, the so-called lipid rafts, in an unsaturated glycophospholipid environment (reviewed in Refs. 6 and 7). Because of their biophysical properties, these cholesterol/sphingolipid rafts are insoluble in nonionic detergent at 4°C and can be isolated as low-density complexes in sucrose gradients. They have also been referred to as detergent-insoluble glycolipid-enriched complexes (8), low-density Triton-insoluble fractions (9), or glycolipid-enriched membrane domains (10). Even though the initial models proposed a central role for lipid rafts in TCR-mediated signaling through mediation of the assembly of complexes, there have been controversies regarding the definition and importance of lipid rafts (11). These controversies were comprehensively discussed in recent reviews (12, 13) where 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. However, in a very recent study the condensation of a plasma membrane at the site of TCR activation was visualized, which confirmed the presence of lipid rafts (14). One important novel contribution of these recent studies has been that protein-protein complexes work in concert with lipid rafts to critically regulate early T cell signaling and the proper formation of the immunological synapse (11, 13, 14).

Several key players in early signal transduction pathways downstream of the TCR, such as the {zeta}-chain of the TCR/CD3 complex, Lck, Fyn, ZAP-70, Shc, linker for activation of T cells (LAT),3 SLP-76, and phospholipase C{gamma}1, have been shown to localize to the lipid raft fraction either constitutively or upon stimulation (10, 15, 16, 17). We have recently demonstrated that in T cell lines as well as in primary thymocytes 20–30% of the protein tyrosine phosphatase Src homology 2 (SH2)-containing phosphatase 1 (SHP-1) constitutively localizes to lipid rafts, which is important for SHP-1 function, and that the level of raft-localized SHP-1 stays constant during T cell activation (18). In contrast with the human polymorphonuclear leukocytes of young donors, the basally lipid raft-localized SHP-1 is temporally displaced upon GM-CSF stimulation, apparently to allow productive signaling (19).

SHP-1 is expressed predominantly in hemopoietic cells of all lineages and all stages of maturation. Based on biochemical, functional, and genetic experiments, the SHP-1 protein and its phosphatase activity have been implicated in the negative regulation of signaling events induced by receptors for Ags, cytokines, and growth factors (reviewed in Refs. 20 and 21). Several lines of data, including analyses of SHP-1 deficient mice, suggest a negative regulatory role for SHP-1 in T cell signaling as evidenced by effects on Lck kinase activation, overall intracellular tyrosine phosphorylation, proliferation, and IL-2 production in response to TCR stimulation (22). SHP-1 is composed of a central catalytic domain, two SH2 domains at its N terminus, and a C terminus with potential tyrosine phosphorylation sites (23). The SH2 domains have been shown to be important for localization (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) as well as for the regulation of SHP-1 catalytic activity (35, 36). In vitro data suggest that the C terminus may also be involved in regulating the phosphatase activity of SHP-1 (reviewed in Ref. 37). In addition to having a regulatory function for enzymatic activity, the C terminus of SHP-1 has also been implicated in containing localization signals (37). A nuclear localization sequence has been identified in the most C-terminal end of SHP-1 that promotes nuclear localization upon cytokine (38) or epidermal growth factor stimulation (39). Our previous work has shown that in T cells the C terminus mediates a constitutive lipid raft localization of SHP-1 (18), although it does not contain any known lipid raft targeting sequences.

In this study, we have identified a novel lipid raft-targeting motif within the C terminus of SHP-1. Mutations of amino acids within this motif result in the loss of lipid rafts localization, whereas fusion of the peptide imparts lipid rafts localization and function to a variant of SHP-1 that cannot by itself localize to lipid rafts. These data indicate that the newly identified 6-mer peptide within the C terminus of SHP-1 is necessary and sufficient for lipid raft localization. Moreover, mutational analyses suggest that lipid raft localization is the primary role of the SHP-1 C terminus when regulating TCR-mediated signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture, plasmids, and generation of stable cell lines

Jurkat T cells (40), parental BYDP murine T cell hybridoma cells (41), and BYDP cells stably expressing hemagglutinin (HA)-tagged SHP-1 were grown in a 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. BYDP cell lines expressing HA-tagged SHP-1 variants were maintained under the selection of 1.0 mg/ml G418. HEK293T were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% FBS, 10 U/ml penicillin, and 10 µg/ml streptomycin.

HEK 293T cells were transfected with derivatives of the pEGFP-C1 vector (BD Biosciences) encoding a fusion between enhanced GFP (EGFP) and full-length or mutant human SHP-1. To transfect the cells, FuGENE 6 transfection reagent (Roche) was used according to the manufacturer’s protocol. Cells were harvested 48–72 h post-transfection. Jurkat cells were transiently transfected with the above pEGFP-C1 constructs using electroporation and harvested 72 h post-transfection. To generate stable cell lines, BYDP cells were transfected by electroporation using derivatives of the pEGFP-C1 vector where the EGFP was replaced by HA-tagged full-length SHP-1 or mutants of SHP-1 followed by selection for G418 resistance. Four to 12 independent clones were isolated for each construct based on expression of the fusion protein.

Different deletion constructs of human SHP-1 were created using standard recombinant DNA techniques. Specifically, the C terminus of SHP-1 was deleted by replacing the codon for aa 527 with a stop codon or an EcoRI site allowing for the fusion to additional coding sequences and subcloned into the pEGFP-C1 vector (thereby replacing EGFP). All of the constructs where peptides of 10 aa or less in length were fused to EGFP or SHP-1 {Delta}C were generated using oligonucleotides (Integrated DNA Technologies) encoding the whole peptide, including a stop codon and EcoRI and BamHI sites for subcloning. Constructs encoding longer peptides were generated by a PCR-based technique. The HA-tagged SHP-1 A6 mutant with six alanine substitutions was created using PCR-based mutagenesis. Bacterial expression plasmids were generated by subcloning the respective constructs into Gex-2T using PCR-based technique. All PCR-based subcloning was confirmed by sequencing.

Stimulation of BYDP cells

BYDP cells were stimulated as described previously (23). Briefly, cells were stimulated through a TCR/CD3{epsilon} complex and CD4 receptors by using 1.0 µg/ml 145-2c11 (anti-CD3{epsilon}) (Southern Biotechnology Associates) in combination with 0.5 µg/ml anti-CD4 (BD Biosciences) followed by cross-linking with 10 µg/ml goat anti-mouse IgG (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 in 0.5 ml of Nonidet P-40 lysis buffer at 4°C (23).

Isolation of lipid rafts

Lipid rafts isolation was performed as described previously (18). Briefly, 6.5 x 107 Jurkat or BYDP cells or HEK 293 cells grown to confluency on 100-mm culture dishes were lysed in 1 ml of 0.5% Triton X-100 lysis buffer at 4°C (18). Cell lysates were mixed with an equal volume of 80% sucrose solution, and the resulting 40% solution was overlaid with 2 ml of 30% sucrose solution and 1 ml of 5% sucrose solution (all sucrose solutions were prepared in buffer (25 mM Tris-Hcl (pH 7.6), 150 mM NaCl, and 5 mM EDTA) containing 0.5% Triton X-100, phosphatase inhibitors (10 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml antipain, and 20 µg/ml PMSF) at 4°C). Samples were subjected to ultracentrifugation in a SW55Ti rotor at 200,000 x g for 20 h at 4°C, and 12 equal fractions of 400 µl each were harvested top to bottom from each gradient. An analysis of each fraction harvested from sucrose gradients was conducted by SDS-PAGE followed by immunoblotting on Immobilon-P polyvinylidene difluoride membrane (Millipore).

To identify detergent-soluble and insoluble fractions, marker proteins were used. For the T cell lines BYDP and Jurkat, LAT (10) and CD45 (42) were chosen as markers for lipid rafts and lipid raft exclusion. Alternatively, for HEK 293 cells the lipid raft marker caveolin (43) and a lipid raft-excluded transmembrane protein, the transferrin receptor (44), were selected as markers. Based on the migration of these marker proteins in the sucrose gradient in our hands and the hands of a number of other investigators (10, 18, 42, 43, 44), fractions 3–5 were identified as lipid rafts and fractions 10 and 11 as detergent soluble. For immunoprecipitations, the respective fractions were subsequently combined and referred to as lipid rafts and detergent-soluble fractions. All paired lipid rafts and detergent-soluble fractions were run on the same gel and exposed for the same length of time.

Immunoprecipitation and immunoblotting

Immunoprecipitation and immunoblotting were performed as previously described (18). Briefly, HA-tagged mutants of SHP-1 were immunoprecipitated from lipid rafts fractions (pooled fractions 3, 4, and 5) or detergent-soluble fractions (pooled fractions 10 and 11) using 2 µg of polyclonal rabbit anti-HA Abs (Santa Cruz). LAT was immunoprecipitated from equal aliquots of Nonidet P-40 lysates of TCR/CD3{epsilon} plus CD4-stimulated cells by using 5 µg of polyclonal rabbit anti-LAT Abs (Upstate Biotechnology).

Immunoblottings were conducted after resolving whole cell lysates, lipid rafts, and detergent-soluble fractions or immunoprecipitates by SDS-PAGE. The primary Abs used were polyclonal rabbit anti-GFP Abs (0.2 µg/ml; Santa Cruz Biotechnology), rabbit anti-caveolin Abs (0.05 µg/ml; BD Transduction Laboratories), mouse anti-CD45 mAb (1/500; BD Transduction Laboratories), mouse anti-transferrin receptor mAb (0.5 µg/ml; Zymed Laboratories), mouse anti-SHP-1 mAb (1.33 µg/ml; Neomarkers), mouse anti-phosphotyrosine mAb (0.5 µg/ml), and mouse anti-LAT mAb (1/2500; Upstate Biotechnology).

Lipid binding assay

To assess direct lipid binding via the C-terminal peptides of SHP-1, a protein-lipid overlay assay was performed using Membrane Lipid Strips (P-6002; Echelon Biosciences) according to the manufacturer’s recommendations. Briefly, membranes were blocked in 3% (w/v) fatty acid-free BSA (Sigma-Aldrich) in TBST (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% (v/v) Tween 20) for 1 h at room temperature in the dark followed by overnight incubation with bacterially expressed and purified GST fusion proteins (1.0 µg/ml in TBST) at 4°C with gentle agitation. After washing the membranes four times over a 30-min period in TBST, they were incubated for 1 h with 1 µg/ml rabbit anti-GST polyclonal Ab (Z-5; Santa Cruz Biotechnology). Membranes were washed as before and incubated for 1 h with anti-rabbit-HRP conjugate (1/10,000 dilution; Pierce) followed by four TBST washes over 1 h and detection of lipid-bound GST fusion proteins by ECL.

IL-2 assay

Amounts of IL-2 production were measured using ELISA as previously described (18). ELISAs were performed using the mouse IL-2 OptEIA ELISA and tetramethylbenzidine substrate reagent set (BD Biosciences) according to the manufacturer’s recommendations. Briefly, 5 x 104 BYDP cells were seeded in 200 ml of complete RPMI 1640 medium in flat-bottom 96-well plates coated with the indicated concentrations of anti-CD3{epsilon} mAb (145-2c11; Southern Biotechnology). As a positive control, PMA (5 ng/ml) and ionomycin (1 mM) were added in uncoated wells. Fifty microliters of supernatants were collected after 24 h of incubation at 37°C and mixed with equal amounts of assay diluent and the OD was measured at 450 nm. Standards of known IL-2 concentrations were included to quantitate the amount of IL-2 in the supernatants.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The C terminus of SHP-1 is necessary and sufficient for lipid raft localization

We have previously shown that in T cell lines and in primary thymocytes 20–30% of total cellular SHP-1 localizes to the detergent insoluble fraction, also referred to as lipid rafts. Moreover, mutants lacking the C-terminal 67 or 76 aa of SHP-1 failed to localize to lipid rafts indicating that this region contains a lipid raft-targeting motif. To test whether the C-terminal 76-aa region of SHP-1 promotes lipid raft localization on its own , we generated fusion proteins between GFP, which fails to localize to lipid rafts by itself, and different subfragments of this 76-aa region of SHP-1 and analyzed their localization to detergent-soluble and insoluble fractions. To confirm that the GFP fusion part does not change the subcellular localization of SHP-1, we first evaluated the localization of a fusion between full-length SHP-1 and GFP (Fig. 1A, top). We observed that the lipid raft localization of GFP-SHP-1 was comparable to what we had previously detected for endogenous SHP-1 in T cells (18). A fusion between GFP and the most C-terminal 76 aa of SHP-1 also localized to lipid rafts (Fig. 1A), indicating that the C terminus of SHP-1 is necessary and sufficient to confer lipid raft localization.


Figure 1
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  		FIGURE 1. The C terminus of SHP-1 is sufficient for lipid raft localization in HEK 293 cells. HEK 293 cells were transfected with the indicated GFP fusion constructs. Transfected cells were lysed in 0.5% Triton X-100 lysis buffer and fractionated via sucrose gradient. Equal aliquots of fractions 3–5 (rafts) and 10 and 11 (detergent soluble), with each fraction corresponding to 8.75% of the total, were separated by 12% SDS-PAGE followed by immunoblotting for GFP. To control for the purity of the lipid raft fractions, all immunoblots were stripped and reprobed for caveolin and transferrin receptor. Data shown is representative of 3–5 independent experiments. A, Raft and non-raft localization of fusions between GFP and full-length SHP-1 or indicated C-terminal fragments of SHP-1. B, Raft and non-raft localization of indicated 6- to 20-aa peptides within the C terminus of SHP-1 fused to GFP.

 
A six-amino acid motif within the C terminus of SHP-1 confers lipid raft targeting

Because there is no known lipid raft-targeting motif within the C terminus of SHP-1, we generated a number of deletions of the C terminus of SHP-1 to further define the lipid raft-targeting domain. We found two overlapping deletion constructs that targeted GFP to the lipid rafts (Fig. 1A). By examining a series of additional shorter fragments within the C terminus of SHP-1, we identified a 6-mer peptide (aa 557–562) as being capable of targeting GFP to lipid rafts (Fig. 1B). Basic Local Alignment Search Tool (BLAST) searches using this 6-mer motif of SHP-1 indicated that this is a unique sequence that is not even found in the closely related phosphatase SHP-2. To further characterize the importance of the residues within this novel lipid raft-targeting motif, we mutated individual amino acids within the 6-mer motif, fused them to the GFP, and tested the ability of these mutants to target to lipid rafts. Interestingly, we observed that all six amino acids are essential for raft targeting via this motif (Fig. 2).


Figure 2
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  		FIGURE 2. Intact SKHKED motif is required for lipid raft localization. HEK 293 cells were transfected with the indicated GFP fusion constructs and their localization to raft and non-raft fractions was analyzed as in Fig. 1 by immunoblotting for GFP. Purity of the lipid raft fractions was confirmed by stripping and reprobing of the blots for caveolin and the transferrin receptor (data not shown). Data shown is representative of three to five independent experiments.

 
To confirm that the above observations made using transient expression in 293T cells were applicable to T cells, we performed two types of analyses. First, we asked whether some of the C-terminal fragments analyzed above can also target the fused GFP moiety to lipid rafts in Jurkat T cells. We confirmed that fragments carrying the 6-mer motif and the 6-mer motif by itself can promote lipid raft targeting in Jurkat cells (Fig. 3).


Figure 3
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  		FIGURE 3. SKHKED motif is sufficient for lipid raft targeting in Jurkat T cells. Jurkat cells were transiently transfected with the indicated GFP fusion constructs. Transfected cells (6.5 x 107) were lysed using 0.5% Triton X-100 and analyzed as in Fig. 1 by immunoblotting for GFP. All immunoblots were stripped and reprobed for LAT and CD45 to control for the purity of lipid raft fractions (data not shown). Data shown are representative of three independent experiments.

 
We next assessed whether the SKHKED motif is functional in the context of a full-length SHP-1 molecule. To test this, we mutated all six amino acids of the SKHKED motif to alanines within the context of full-length SHP-1 and generated BYDP murine T cell hybridoma cells stably expressing this construct (HA-SHP-1 A6). We compared BYDP cells stably expressing HA-SHP-1 with HA-SHP-1 A6. Although a fraction of the HA-SHP-1 localized to the lipid rafts as previously shown, the HA-SHP-1 A6 mutant failed to localize to the detergent-insoluble fractions (Fig. 4), indicating that the SKHKED motif is essential for lipid raft localization of full-length SHP-1. This also suggested that other residues within the C-terminal 67 aa of SHP-1 (the fragment initially identified as important for raft targeting) cannot substitute for the SKHKED motif.


Figure 4
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  		FIGURE 4. The SKHKED motif is required for raft localization in the context of full-length SHP-1 in BYDP T cell hybridomas. BYDP cells (6.5 x 107) stably expressing HA-tagged full-length (HA-SHP-1) or mutant SHP-1 (HA-SHP-1 A6 that contains six alanines replacing the SKHKED motif) were lysed in 0.5% Triton X-100 lysis buffer and fractionated via sucrose gradient. HA-fusion proteins were immunoprecipitated from equal aliquots (75% of each fraction) of combined lipid rafts (3 4 5 ) and detergent-soluble (10 11 ) fractions, followed by electrophoresis using 10% SDS-polyacrylamide gels and immunoblotting for SHP-1.

 
We next asked whether the addition of the SKHKED motif to SHP-1 {Delta}C, a mutant that lacks the last 67 aa and fails to localize to lipid rafts, can now restore lipid raft targeting. We generated expression constructs where we fused SHP-1 {Delta}C with either the SKHKED motif, a mutation of this motif where the two lysines are mutated to alanine (denoted SAHAED), or a C-terminal fragment lacking the SKHKED motif (aa 575–595). Upon stable expression of these constructs in the BYDP T cell hybridoma cell line, we tested their lipid rafts localization. Although the addition of the 6-mer SKHKED motif restored lipid raft targeting to the SHP-1 {Delta}C, the fusion of SAHAED or the 575–595 sequence failed to restore lipid raft targeting (Fig. 5). Taken together, these data identified the SKHKED motif as a novel and important component of the SHP-1 C terminus that is necessary and sufficient for targeting this phosphatase to lipid rafts.


Figure 5
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  		FIGURE 5. The SKHKED motif is sufficient for raft localization of SHP-1 in BYDP cells. BYDP cells (6.5 x 107) stably expressing HA-tagged wild-type SHP-1, SHP-1 {Delta}C, or SHP-1 {Delta}C fused to the indicated C-terminal peptides of SHP-1 were lysed in 0.5% Triton X-100 and analyzed as in Fig. 4 by anti-HA immunoprecipitation followed by immunoblotting for SHP-1. The depicted constructs are not drawn in scale, but the C terminus is enlarged compared with the rest of the protein to show more details.

 
Because this motif has not been previously identified as a lipid raft-targeting motif, we chose to examine the underlying targeting mechanism. To address whether C-terminal peptides that contain the 6-mer motif might directly bind to membrane lipids, protein-lipid overlay assays were performed using bacterially expressed GST-C-terminal peptide fusion proteins. Analysis of a 545–573 peptide (GST plus 545–573), which encompasses the 6-mer motif, showed preferred binding to phosphatidic acid and phosphatidylinositol 4-phosphate, and, to a lesser extent, binding to phosphatidylinositol 4,5-bisphosphate, and phosphatidylserine (Fig. 6). In contrast, a mutant of the 545–573 peptide (GST plus 545–573 A6) where all six amino acids of the motif had been mutated to alanines failed to bind any of the lipids. These data suggest that the 6-mer motif in SHP-1 can directly bind phospholipids, providing a potential mechanism for lipid raft targeting of SHP-1.


Figure 6
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  		FIGURE 6. Phospholipid binding of the C-terminal SHP-1 peptide is dependent on intact SKHKED motif. The indicated fusion proteins (1 µg/ml) were incubated with membrane lipid strips followed by the detection of bound protein. GST protein by itself does not bind to any of the lipids (data not shown). Template on the left depicts which lipids are loaded onto the strip and indicates the preferred lipid binding of the C-terminal peptide. DAG, 1,2-Diacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate.

 
The SKHKED motif is essential for SHP-1 mediated regulation of TCR signaling

SHP-1 is well recognized as a negative regulator of TCR-mediated signaling. We have previously demonstrated that the expression of full-length SHP-1 in BYDP cells inhibits signal transduction pathways downstream of the TCR by using both biochemical and downstream functional readouts (18). Specifically, TCR cross-linking of cells expressing full-length SHP-1 failed to lead to LAT or {zeta}-chain tyrosine phosphorylation. Moreover, SHP-1-expressing cells produced greatly reduced levels of IL-2 in response to TCR/CD3 stimulation. In contrast, T cells expressing the catalytically active but raft targeting-impaired SHP-1 {Delta}C mutant did not inhibit TCR/CD3-mediated signaling. Although our data suggested that the failure to localize to lipid rafts might be the cause of this inactivity, it did not exclude the possibility that the C terminus harbors other elements critical for its function as a negative regulator. Our identification of the SKHKED motif in the studies above allowed us to address the importance of the SHP-1 C terminus with respect to raft targeting vs other functions. We compared the functional responses of BYDP cells stably expressing HA-SHP-1or the HA-SHP-1 A6, SHP-1 {Delta}C, SHP-1 {Delta}C plus SKHKED, and SHP-1 {Delta}C plus (575–595) variants.

Upon TCR/CD3 plus CD4 stimulation, BYDP cells (parental line) show an increase in overall tyrosine phosphorylation (Fig. 7A) as well as inducible LAT tyrosine phosphorylation (Fig. 7C). In HA-SHP-1-expressing cells this inducible phosphorylation was inhibited. However, expression of the HA-SHP-1 A6 mutant, which fails to localize to lipid rafts (Fig. 4), did not inhibit the TCR/CD3-mediated total tyrosine phosphorylation (Fig. 7A) or LAT tyrosine phosphorylation (Fig. 7C). Remarkably, the addition of the SKHKED motif restored functionality to the SHP-1 {Delta}C mutant and was able to inhibit both total tyrosine phosphorylation and LAT phosphorylation induced byTCR/CD3 cross-linking (Figs. 7, A and C).


Figure 7
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  		FIGURE 7. The SKHKED motif is necessary and sufficient for the inhibition of TCR-mediated tyrosine phosphorylation by SHP-1. Parental or HA-tagged SHP-1 variants expressing BYDP cells (2 x 107) were stimulated by TCR/CD3{epsilon} cross-linking for the indicated times and lysed in 1% Nonidet P-40 lysis buffer. A, Equal aliquots (5%) of cell lysates were separated by electrophoresis with a 10% SDS-polyacrylamide gel and immunoblotted with and anti ({alpha})-phosphotyrosine (pTyr) Ab. To control for equal loading, immunoblots were stripped and reprobed for LAT. B, To confirm comparable protein expression between different clones, HA-tagged SHP-1 fusion proteins were immunoprecipitated (IP) from cell lysates (3.2 x 107 cells) using anti-HA Abs followed by immunoblotting for SHP-1. C, LAT was immunoprecipitated from equal amounts (60%) of cell lysates using anti ({alpha})-LAT Abs followed by immunoblotting for tyrosine phosphorylation (pTyr). Immunoblots were stripped and reprobed for LAT. These data are representative of experiments that were repeated three times with at least two independent clones.

 
As a functional downstream readout, we examined IL-2 production by the same cell lines in response to TCR/CD3 stimulation. Although parental BYDP cells generate IL-2 in response to increasing concentrations of anti-CD3 Ab, IL-2 production was inhibited by the expression of full-length SHP-1. In contrast as we had shown previously, the lipid raft-excluded HA-SHP-1 {Delta}C mutant failed to inhibit IL-2 production. However, lipid raft targeting of this SHP-1 {Delta}C mutant via the SKHHED motif restored the ability of SHP-1 to inhibit TCR-mediated IL-2 production (Fig. 8A). As a parallel analysis, we also addressed whether the full-length SHP-1 in which the SKHKED motif has been mutated to alanine can affect TCR-mediated IL-2 production. This raft-excluded HA-SHP-1 A6 mutant failed to inhibit TCR-mediated IL-2 production (Fig. 8B). These data indicate that the primary function of the C terminus of SHP-1 is raft targeting via the SKHKED motif and that such targeting is critical for this phosphatase to act as a negative regulator in T cells.


Figure 8
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  		FIGURE 8. The SKHKED motif is necessary and sufficient for inhibition of IL-2 production by SHP-1. Cells (5 x 104) of the indicated cell lines were stimulated with increasing concentrations of plate-bound anti-CD3{epsilon} Ab. Supernatants were collected after 24 h and analyzed for IL-2 content by ELISA. All experiments were performed at least three times in triplicate and the data shown are averages. Error bars represent the SD of the mean. A, Parental BYDP cells or BYDP cells stably expressing HA-tagged full-length SHP-1, SHP-1 {Delta}C, or lipid raft-targeted SHP-1 {Delta}C plus SKHKED. B, BYDP cells stably expressing HA-tagged full-length SHP-1 or SHP-1 A6.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies have suggested that the lipid raft targeting of SHP-1 is functionally important for this tyrosine phosphatase to regulate early TCR-mediated phosphorylation events and to regulate downstream events such as IL-2 production (18, 45, 46). It has also been shown that SHP-1 mutants lacking the C-terminal 67 aa failed to localize to lipid rafts and demonstrated impaired SHP-1 function. However, how these C-terminal 67 residues regulate the targeting of SHP-1 to rafts or whether this is the primary function of this region was unknown. Notably, the C terminus of SHP-1 lacks any of the previously defined motifs known to promote lipid raft localization; although the C terminus has been shown to bind to phospholipids (47) such as phosphatidic acid, these lipids are supposed to be enriched outside lipid rafts. Thus, the role of the C terminus in SHP-1 function and how it mediated raft targeting were puzzling. In this study we made a series of observations that led to the identification of a novel six-amino acid SKHKED motif as necessary and sufficient for lipid raft targeting of SHP-1 and for SHP-1 function.

This SKHKED peptide motif identified in SHP-1 has not been previously recognized for its ability to target proteins to lipid rafts, suggesting that this is a novel lipid raft-targeting motif. Surprisingly, a search of the eukaryotic protein databases failed to identify other lipid raft-localized proteins containing the same motif. Moreover, this motif seen in SHP-1 is not seen in the closely related SH2 domain-containing tyrosine phosphatase SHP-2. Whether variations of this motif capable of promoting lipid raft localization exist in other proteins remains to be seen. However, it is notable that our mutational analyses also demonstrated that the 6-mer motif is the minimal targeting sequence and that any nonconservative mutation within this motif abrogates lipid raft localization.

Because previous studies only showed the requirement of the 67-aa C-terminal sequence in targeting SHP-1 to lipid rafts, it was not known whether one or more mechanisms of targeting exist within this sequence. Our studies described in this article, where we mutated just the SKHKED motif to alanine in the context of full-length SHP-1 or fused this motif to the SHP-1 {Delta}C mutant, suggested that the SKHKED is the only mode by which the C terminus of SHP-1 mediates lipid raft targeting. Because this 6-mer peptide is a previously unrecognized targeting motif, the underlying mechanism of its membrane targeting was not known. Using purified bacterial proteins, we observed direct lipid binding via the C-terminal peptide, preferentially toward phosphatidic acid, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate, whereas mutation of the 6-mer motif abolished this binding activity. Because phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate have previously been shown to be enriched in lipid rafts (48, 49), these data suggest that the 6-mer motif mediates lipid raft targeting via direct lipid binding. It has been previously reported that the last 41 aa of SHP-1, which includes the 6-mer peptide, are able to bind to phosphatidic acid and in particular phosphatidic acid-containing saturated fatty acids, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (50). However, that study did not address whether binding to phospholipids might affect the subcellular localization of SHP-1. In the present study we show that a shorter fragment mediates binding to phosphatidic acid and phosphatidylinositol 4,5-bisphosphate, but not to phosphatidylinositol 3,4,5-trisphosphate, and that this binding is dependent on an intact 6-mer motif that is able to mediate lipid raft localization. Interestingly, saturated fatty acids, which SHP-1 seems to bind with preference (50), have been shown to be enriched in lipid rafts compared with the rest of the plasma membrane (51, 52). Although we have shown that a peptide containing the targeting motif can bind to phospholipids that are enriched in the lipid rafts, future studies will have to address whether this binding is mediating both the targeting and the retaining of SHP-1 to lipid rafts or whether the lipid binding mostly serves to target SHP-1 to lipid rafts and whether additional forces, such as protein-protein interactions, are required to sustain the lipid raft localization. It is, for example, possible that the recently discovered raft targeting scaffolding proteins, such as Dlgh1 (53) are involved in maintaining SHP-1 in the lipid rafts. However, to date we have been unable to detect any stable protein-protein interactions of lipid raft-localized SHP-1 with other proteins (M. Sankarshanan and U. Lorenz, unpublished data). We recognize that such a negative result does not rule out any association of SHP-1 with other proteins, as it is possible that such complexes might not sustain cell lysis conditions.

It has previously been shown that the most C-terminal region of SHP-1 contains a bipartite nuclear localization signal that mediates nuclear localization of SHP-1 in epithelial cells (38, 54). However, as shown in our present studies, this sequence encompassing aa 576–595 is dispensable for lipid raft localization and the regulatory function of SHP-1 in T cells. Whether SHP-1 might have additional functions in epithelial cells and whether a lipid raft-excluded mutant of SHP-1 that still retains the ability to localize to the nucleus might reveal other functions of SHP-1 in epithelial cells remains to be seen.

Taken together, in this report we have identified a novel lipid raft-targeting SKHKED motif that is necessary and sufficient to promote lipid raft localization of SHP-1. In addition to the critical role it plays for SHP-1 function, this motif could potentially constitute a tool for targeting proteins of choice to phospholipids and in particular to lipid rafts. Moreover, mutations within the motif render it inactive, providing the necessary control. Our data suggest that in T cells, where SHP-1 has been shown to be a negative regulator of TCR-mediated signaling, lipid raft localization is absolutely required for SHP-1 function and that the primary role of the C terminus of SHP-1 is in guiding this subcellular localization.


   Acknowledgments
 
We are grateful to members of our laboratory and to Dr. Kodi Ravichandran for critical reading of the manuscript, comments, and suggestions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 R01 AI48672 and an award from the Thomas F. and Kate Miller Jeffress Memorial Trust. Back

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. E-mail address: ulorenz{at}virginia.edu Back

3 Abbreviations used in this paper: LAT, linker for activation of T cells; EGFP, enhanced GFP; HA, hemagglutinin; SH2, Src homology 2; SHP-1, SH2-containing phosphatase 1. Back

Received for publication December 19, 2006. Accepted for publication April 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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