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International Journal of Clinical and Experimental Medicine logoLink to International Journal of Clinical and Experimental Medicine
. 2015 Apr 15;8(4):6568–6577.

T cell activation is reduced by the catalytically inactive form of protein tyrosine phosphatase SHP-2

Baoxia Dong 1,*, Yubo Gao 2,*, Xuan Zheng 1, Guangxun Gao 1, Hongtao Gu 1, Xiequn Chen 1, Jinyi Zhang 3
PMCID: PMC4483852  PMID: 26131287

Abstract

Src-homology 2 domain-containing tyrosine phosphatase-2 (SHP-2) is a ubiquitously expressed cytosolic tyrosine phosphatase implicated in many different signaling pathways involving cytokine receptors and T and B cell receptors; however, the precise functional role of SHP-2 in T cell signaling is not entirely clear. In this study, we overexpressed a catalytically inactive form of SHP-2 with a classic cysteine 459-to-serine mutation (dnSHP-2) to elucidate the in vivo effects of SHP-2 on T cells. We found that mice overexpressing dnSHP-2 showed reduced T cell activation, presumably due to increased tyrosine phosphorylation of Grb2-binding protein (Gab2) and inhibition of mitogen-activated protein kinase (MAPK) activity. SHP-2 appears to be a positive regulator of the MAPK pathway in T cells, likely through coupling of the multimeric complex to the Ras/MAPK pathway. However, SHP-2 does not appear to affect T cell antigen receptor (TCR)-evoked calcium mobilization, stress-activated protein kinase/c-jun N-terminal kinases (SAPK/JNKs) activation, or overall tyrosine phosphorylation.

Keywords: Src-homology 2 domain-containing tyrosine phosphatase-2, Gab2, Ras/MAPK pathway, T cell, transgenic mice

Introduction

SHP-2 is a ubiquitously expressed 70-72-kDa cytosolic tyrosine phosphatase that is implicated in many different signaling pathways [1,2]. It is a member of a subfamily of protein tyrosine phosphatases that possess two src-homology 2 (SH2) domains in the N-terminus. SHP-2 was found to associate with various receptor protein tyrosine kinases, including the insulin receptor, the platelet-derived growth factor (PDGF) receptor, the epidermal growth factor (EGF) receptor, and the erythropoietin receptor [3-5]. It also associates with adaptor molecules, such as insulin receptor substrate-1 (IRS-1) and Grb2, transmembrane proteins, such as SH2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1), and adhesion molecules such as platelet/endothelial cell adhesion molecule 1 (PECAM-1) [6-9]. It is implicated in signaling pathways involving cytokine receptors and T and B cell receptors [10-12].

SHP-2 is heavily phosphorylated on tyrosyl residues by receptors and transforming protein tyrosine kinases, and this raises the question of whether it can also act as an adaptor protein [13]. Indeed, upon stimulation of the platelet-derived growth factor (PDGF) receptor, which associates with SHP-2, was shown to bind Grb2 at three potential sites. This finding led to the suggestion that SHP-2 acts as an adaptor between the PDGF receptor and the Grb2-Sos complex [14]. Another study suggested that SHP-2 functions as an adaptor between the activated c-kit receptor and Grb2 [15].

In most cases, SHP-2 is a positive regulator of a variety of signal transduction pathways. It is required for Ras-MAPK cascade activation; expression of a dominant negative form of SHP-2 inhibited MAPK activation in response to various signals, including insulin and fibroblast growth factor (FGF) [2,16]. The exact point where SHP-2 acts in the signaling pathways was not entirely clear. Some studies have placed it upstream of MAPK, and perhaps even upstream of Ras [17]. The effects of SHP-2 on jun N-terminal kinase (JNK) activity are also somewhat unclear, although it was found to be required for insulin-induced JNK activation [18]. However, it was also shown that SHP-2 was a negative effector of cellular stress-induced JNK activation in mouse embryonic fibroblast cells [19]. Another recent study demonstrated that SHP-2 is a negative regulator of the interferon-stimulated JAK/STAT pathway [10]. Moreover, SHP-2 is also involved in the control of cell adhesion, migration, and cytoskeletal architecture [20]. It was recently shown that activation of the anti-apoptotic kinase Akt was reduced in SHP-2-deficient fibroblasts [21].

Although previous results demonstrate that SHP-2 is important for development and growth factor signaling, little is known regarding its role in T cells. A deletion mutation in the SH2 domain severely suppresses hematopoietic cell development [22], and chimeric mice with Shp-2 mutant sells also show suppressed hematopoiesis and multiple development defects [23]. Homozygous deletion of SHP-2 in mice leads to embryonic lethality at mid-gestation [14], so we generated a transgenic mouse line that overexpressed a dominant negative, catalytically inactive form of SHP-2 in T cells to identify its effect on T cell activation.

Materials and methods

The study was approved by the Animal Care and Use Committee of Xijing Hospital.

Generation of dnSHP-2 transgenic mice

We generated transgenic mice that overexpress a catalytically inactive form of SHP-2 specifically in T cells. This work involved subcloning a FLAG-tagged, murine SHP-2 cDNA carrying the classic cysteine 459-to-serine mutation in the phosphatase domain (dnSHP-2) into the T cell-specific vector p29Δ2 (sal-) that contains a CD2 promoter and enhancer (Figure 1). The transgene fragment was purified and microinjected into CD1 zygote pronuclei. Initial genotyping to identify the transgene- bearing founders was done by Southern blotting. The founders were bred on a C57BL/6 background. Subsequent genotyping to identify mice bearing the transgene was done by polymerase chain reaction (PCR). The studies described below were performed in mice that were backcrossed over three generations to the C57BL/6 background.

Figure 1.

Figure 1

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Generation of dnSHP-2 transgenic mice. Subcloning of a FLAG-tagged, murine SHP-2 cDNA carrying the classic cysteine 459-to-serine mutation in the phosphatase domain into the T cell-specific vector p29D2 (sal-). Western blotting with SHP-2 antibodies of lysates from the LNs and thymus of WT and transgenic mice.

Flow cytometry

Flow cytometry was performed using a FACScalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Antibodies to the following cell surface markers were used (all BD Pharmingen/BD Biosciences, unless stated otherwise): CD3f (145-2C11; in-house hybridoma), CD4 (YTA3.1.2; in-house), CD8 (53.6.7), CD25 (PC61), CD44 (IM7), B220 (RA3-6B2), CD69 (H1-2F3), and DO11.10 T cell antigen receptor (TCR, KJ-126). For secondary labeling, fluorescein isothiocyanate (FITC-), phycoerythrin (PE-), or allophyocyanin-conjugated secondary antibodies were used. At least 10,000 events were captured and analyzed using CellQuest software (BD Biosciences).

Cell proliferation assay

We performed preliminary experiments using the F3 generation mice. Thymocytes and lymph node T cells from the dnSHP-2 transgenic mice and their wild-type (WT) littermates were cultured in 96-well flat-bottomed microtiter plates for 48 hours in culture medium alone or in the presence of various concentrations of soluble CD3 antibody (2C11) alone, CD3 antibody and CD28 antibody (H57), cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody and secondary anti-hamster IgG, phorbol 12-myristate 13-acetate (PMA) plus ionomycin, or Con A (all above from Sigma, St. Louis, MO, USA). Thymocytes were also cultured with interleukin-2 (IL-2) (Sunnybrook Hospital, Toronto, Ontario, Canada). The cells were then pulsed with [3H] thymidine for 16 hours. Incorporated radioactivity was measured using an automated b scintillation counter.

Measurement of intracellular calcium flux

A total of 7 × 106 LN cells from WT or dnSHP-2 transgenic mice were rested in complete medium at room temperature for 1 h. Cells were loaded with 5 g/ml cell-permeable Indo-1 (Molecular Probes/Invitrogen, Carlsbad, CA, USA) for 30 min at 37°C. Cells were then washed and incubated with a biotinylated anti-TCR antibody at 4°C. After washing, the cells were resuspended and stimulated with streptavidin. Calcium levels were detected by flow cytometric analysis of the Indo-1 violet-blue fluorescence ratio.

MAPK activation assay

Lysates from unstimulated and stimulated thymocytes were immunopreciptated with a mixture of anti-ERK1 and anti-ERK2 antibodies. The ERK1/ERK2 immune complexes were washed in MAPK buffer and resuspended in reaction buffer containing, among other components, myelin basic protein (MBP) and [g-32P] ATP. After incubation, the reaction was terminated and the samples were boiled, electrophoresed through SDS-PAGE gel, and transferred to nitrocellulose membrane. The phosphorylated MBP bands were then visualized by autoradiography.

Immunoprecipitation and immunoblotting procedures

A total of 107 LN cells from WT or dnSHP-2 transgenic mice were lysed in lysis buffer (20 mM Tris/HCl, pH 8.0; 1% NP-40; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 10 g/ml aprotinin; 10 g/ml leupeptin; and 10 M Na3VO4) at 4°C. The lysates (1 mg of protein) were clarified by centrifugation at 13,000 × g for 10 min and immunoprecipitated with the appropriate Ab. Immunoprecipitates were washed three times with lysis buffer, then boiled with sodium dodecyl sulfate (SDS) sample buffer for 10 min, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore, Billerica, MA, USA), followed by detection with the appropriate Ab as described previously [24].

Results

T cell development in the dnSHP-2 transgenic mice

To confirm the generation of the transgenic mice, western plotting was performed for the protein in the thymus and LNs of the transgenic mice with anti-FLAG and anti-SHP-2 antibodies (Figure 1). The total cell numbers of the thymocytes and LN T cells of the transgenic mice and their WT littermates were determined by cell counting using a hemacytometer. Our results showed that there was a decrease in the cell numbers of the thymus of dnSHP-2 mice compared with their WT littermates (Figure 2). The results from fluorescence-activated cell sorting (FACS) analyses showed no significant difference in thymocyte maturation between the transgenic mice and their WT littermates. Relative numbers of single- and double-positive thymocytes were similar in the transgenic mice and their WT littermates (Figure 2). Transgenic mice expressed normal levels of maturation markers, including CD5, CD44, and CD25 (data not shown).

Figure 2.

Figure 2

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Immunofluorescence analysis of thymocyte development in dnSHP-2 transgenic mice. Thymocytes from dnSHP-2 transgenic mice (dnSHP-2) and WT littermates were assayed for CD4 and CD8 expression (left panels) or TCRβ chain and CD3ε expression (right panels). Numbers above the left panel indicate total number of thymocytes, and numbers in the quadrants represent percentages of different cell populations.

Reduced antigen receptor-evoked T cell proliferation in dnSHP-2 mice

Our data from F3 mice revealed that proliferation measured by [3H] thymidine incorporation was reduced in both the thymocytes and LN T cells of dnSHP-2 transgenic mice upon stimulation with a CD3 antibody and co-treatment with CD3 and CD28 antibodies (Figure 3). A similar outcome was observed when these cells were stimulated with Con A, but there was no difference in the proliferative response to PMA and ionomycin between the transgenic mice and their WT littermates (Figure 3). When LN T cells were co-cross-linked with a CTLA-4 antibody along with CD3 and CD28, the CTLA-4 mediated inhibition of proliferation was normal in dnSHP-2-expressing T cells (Figure 3). These data suggested that SHP-2 is a positive regulator of TCR signaling functions. However, SHP-2 did not appear to play a role in the negative regulatory functions of CTLA-4 in TCR signaling.

Figure 3.

Figure 3

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Effects of dnSHP-2 on antigen receptor-evoked proliferation. Thymocytes and LN T cells of dnSHP-2 transgenic mice (dnSHP-2) and their WT littermates were cultured for 48 hours with a CD3 antibody, CD3 plus CD28 antibodies, CD3 plus CD28 plus CTLA-4 antibodies (LN T cells only), PMA and ionomycin, or Con A. Proliferative responses were determined after a 16-hour pulse with [3H] thymidine.

SHP-2 involvement in various TCR-evoked signaling functions

Antigen receptor-induced MAPK activation is attenuated in dnSHP-2 thymocytes. To understand the molecular basis for the observed T cell proliferation defects, we first assessed the extracellular signal-related kinase (ERK) signaling pathway. Total thymocytes freshly obtained from animals were stimulated with CD3 plus CD28 antibodies followed by cross-linking with anti-hamster IgG in vitro, and protein extracts were immunoblotted with ERK1 and ERK2 antibodies and control IgG. Although a significant induction of phosphorylated myelin basic protein (p-MBP) signal was observed at 2, 5, and 10 min in control cell lysates, p-MBP levels were dramatically decreased at these time points in dnSHP-2 mice following anti-CD3 or anti-CD28 treatment (Figure 4). These results demonstrate that SHP-2 has a positive role in mediating TCR-triggered ERK activation in thymocytes, which is consistent with an observation made by many groups who found that SHP-2 acts to promote signaling through the ERK pathway in a variety of cell types [25-27].

Figure 4.

Figure 4

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Effects of dnSHP-2 on MAPK activation. Thymocytes from dnSHP-2 transgenic mice and their WT littermates were stimulated with CD3 plus CD28 antibodies followed by cross-linking with anti-hamster IgG for the indicated times. Lysates were immunoprecipitated with ERK1 and ERK2 antibodies, as well as control IgG (C). The immune complexes were evaluated for their ability to phosphorylate MBP by SDS-PAGE and autoradiography (upper panel) and for ERK2 levels by immunoblotting analysis (bottom panel).

No difference in antigen receptor-induced overall tyrosine phosphorylation or calcium mobilization between dnSHP-2 and Wtransgenic mice

As SHP-2 is a cytosolic tyrosine phosphatase implicated in many different signaling pathways, we attempted to determine its effects on overall tyrosine phosphorylation and calcium mobilization signaling. Figure 5 shows that similar levels of anti-CD3 and CD28-stimulated overall tyrosine-phosphorylated proteins were detected in transgenic mice and their WT littermates (Figure 5, left panel). CD3 stimulation of intracellular calcium mobilization was assessed by flow cytometry. No significant different in TCR-evoked calcium influx was seen between the transgenic mice and their WT littermates in preliminary experiments (Figure 5, right panel).

Figure 5.

Figure 5

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Effects of dnSHP-2 on tyrosine phosphorylation and calcium mobilization. Left panel: Thymocytes from dnSHP-2 transgenic mice and their WT littermates were stimulated with CD3 plus CD28 antibodies followed by cross-linking with anti-hamster IgG for the indicated times. Lysates were resolved over SDS-PAGE and subjected to immunoblotting analyses with a phosphotyrosine antibody. Right panel: Flow-cytometric analysis of calcium influx after stimulation of thymocytes with a TCR antibody. Arrows indicate addition of cross-linking streptavidin.

No difference in antigen receptor-induced SAPK/JNK activation between dnSHP-2 and Wtransgenic mice

SHP-2 is required for the activation or negative regulation of JNK in different cell types [10,18,19]. However, the precise effects of SHP-2 on JNK activity are somewhat unclear. We assessed SAPK/JNK activation following stimulation with CD3 plus CD28 antibodies for different time points by western blot. Although a significant induction of phosphorylated SAPK/JNK was observed at 2, 5, and 10 min in both WT and dnSHP-2 cell lysates, no significant difference between the groups were found at these time points in response to anti-CD3 or anti-CD28 treatment (Figure 6).

Figure 6.

Figure 6

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Effects of dnSHP-2 on SAPK/JNK activation. Thymocytes from dnSHP-2 transgenic mice and their WT littermates were stimulated with CD3 plus CD28 antibodies followed by cross-linking with anti-hamster IgG for the indicated times. Lysates were resolved over SDS-PAGE and subjected to immunoblotting analyses with a phospho-SAPK/JNK antibody (top panel) and a JNK (JNK1 and JNK2) antibody (bottom panel).

Association and tyrosine phosphorylation status of possible SHP-2-interacting proteins

As we know, SHP-2 forms a multimeric cytosolic complex with many molecules, such as phosphoinositide 3-kinase (PI3K), zeta-chain-associated protein kinase 70 (ZAP-70) and TCR-ζ, which may provide a link between SHP-2 and Ras/MAPK pathway activation [11]. To determine protein association and tyrosine phosphorylation status of proteins that may interact with SHP-2, lysates from unstimulated and stimulated thymocytes from dnSHP-2 transgenic mice and their WT littermates were immunoprecipitated with a p85 PI3K antibody, a ZAP-70 antibody, or a TCR-ζ antibody. Our data revealed hyperphosphorylation of p36 and p110 proteins that were associated with p85 PI3K in dnSHP-2 transgenic mice, but no affect on the hyperphosphorylation of PI3K itself (Figure 7). These hyperphosphorylation proteins had the same sizes as linker for activation of T cells (LAT) and Gab2. When immunoprecipitation with ZAP-70 or TCR-ζ antibodies were performed followed by immunoblotting with a phosphotyrosine antibody, there was no change in the tyrsoine phosphorylation of ZAP-70 or TCR-ζ in dnSHP-2 transgenic mice (Figure 8).

Figure 7.

Figure 7

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Effects of dnSHP-2 on PI3K-associated proteins. Thymocytes from dnSHP-2 transgenic mice and their WT littermates were stimulated with CD3 plus CD28 antibodies followed by cross-linking with anti-hamster IgG for the indicated times. Lysates were subjected to immunoprecipitation with a p85 PI3K antibody, as well as control rabbit IgG (C), resolved over SDS-PAGE, and subjected to immunoblotting analyses with a phosphotyrosine antibody (top panel) and a p85 PI3K antibody (bottom panel).

Figure 8.

Figure 8

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Effects of dnSHP-2 on ZAP-70 and CD3ζ. Thymocytes from dnSHP-2 transgenic mice (dnSHP-2) and their WT littermates were stimulated with CD3 and CD28 antibodies followed by cross-linking with anti-hamster IgG for the indicated times. Lysates were subjected to immunoprecipitation with a ZAP-70 antibody (top panel) or CD3ζ antibody (bottom panel), as well as control rabbit IgG (C), resolved over SDS-PAGE, and subjected to sequential immunoblotting analyses with phosphotyrosine and ZAP-70 antibodies (top panel) or a CD3ζ antibody (bottom panel).

Discussion

SHP-2 is known to play a vital role in mammalian development and signaling pathways. As mentioned above, SHP-2 knockout is embryonic lethal, and attempts to generate mice that lack SHP-2 using a recombination-activating gene (RAG) knock-out system have also been unsuccessful. In this study, we addressed the role of SHP-2 in T cells using transgenic mice expressing a dominant-negative version of the protein. Characterization of the mice confirmed that the catalytically inactive form was expressed specifically in T cells and that it functioned to inhibit endogenous SHP-2 activity.

SHP-2 is known to be a positive regulator of MAPK activation in a number of systems, and MAPK is involved in thymocyte differentiation [28,29]. In our study, we found that overexpression of the catalytically inactive form of SHP-2 (dnSHP-2) reduced T cell activation but not maturation. The data obtained in this study support the notion that SHP-2 acts to promote T cell maturation/proliferation at both the double-negative (DN) and double-positive (DP) cell stages. The SHP-2-/- transgenic mice exhibited high efficiency and fidelity of SHP-2 gene deletion in thymocytes, mediated by the Cre recombinase, which is consistent with previous reports using this Cre transgenic mouse line [30]. The SHP-2 deletion significantly suppressed progression of DN3 thymocytes to the DN4 stage, as evidenced by DN3 cell accumulation and decreased DN4 cell numbers. The reduced DN cell number in the S and G2 phases of the cell cycle also illustrate a role of SHP-2 in amplifying proliferative signals emanating from the pre-TCR. Thus, the SHP-2 mutation impairs thymocyte differentiation and proliferation by affecting the critical selection step instructed by the pre-TCR signals, ultimately leading to reduced thymic cellularity in SHP-2-/- transgenic mice.

As described above, dnSHP-2 overexpression in Jurkat T cells inhibited TCR-evoked MAPK activation. In our study, reduced MAPK activation in dnSHP-2 thymocytes confirmed that the effect of SHP-2 on MAP kinase is one of the mechanisms of regulating T cell development. SHP-2 forms a multimeric complex with Gab2, Grb2, and PI3K upon TCR stimulation [31], and it has been suggested that SHP-2 activates MAPK by de-phosphorylating Gab2, leading to the release and/or activation of PI3K from the complex. Recently, Akt activation was shown to be reduced in SHP-2-deficient fibroblasts, and this situation may also occur in T cells overexpressing dnSHP-2 in view of the above hypothesis that PI3K, which is involved in Akt activation, takes part in the above-mentioned multimeric complex that also includes SHP-2. Other TCR-evoked signaling functions, including PI3K activation, SAPK/JNK activation, and apoptosis, were not studied in Jurkat T cells. Also, in view of the suggestion that the association of SHP-2 with CTLA-4 may play a role in the inhibition of T cell activation [32-35], it will be useful to determine the response of T cells that overexpress dnSHP-2 to CTLA-4-mediated inhibition. Therefore, we employed the dnSHP-2 transgenic mice to determine the effects of SHP-2 on various TCR signaling pathways in vivo. Data obtained in this study support the notion that SHP-2 acts to promote MAPK as one of the regulatory mechanisms of T cell development. However, SHP-2 does not appear to affect TCR-evoked calcium mobilization, SAPK/JNK activation, or overall tyrosine phosphorylation.

SHP-2 is a ubiquitous phosphatase containing SH2 domains that plays major biological functions in response to various growth factors, hormones, and cytokines. This is essentially due to its particularity in promoting Ras/MAPK pathway activation. Recent progress has been made in the understanding the molecular mechanisms involved in this regulation. There are several different mechanisms explaining the positive role of SHP-2 in MAPK pathway activation. Once recruited in the vicinity of the receptor tyrosine kinase (RTK, direct binding or through an adaptor protein), SHP-2 can be phosphorylated by the RTK, then act as an adaptor protein for mobilizing Grb2/SOS. Alternatively, SHP-2 can dephosphorylate several targets, which are dephosphorylated by the RTK, then act as an adaptor protein for mobilizing Grb2/SOS. SHP-2 can also phosphorylate several targets, the dephosphorylation of which will promote MAPK activation. These include tyrosine phosphatase non-receptor type substrate-1 (SHPS-1) and major vault protein (MVP), for which the precise connections with the MAPK pathway remain to be elucidated, and Sprouty protein (Spry), RasGAP, and Src, for which more precise mechanisms have been proposed. Indeed, SHP-2 can dephosphorylate Spry, which, when phosphorylated, sequesters Grb2/Sos in the cytoplasm [36]. SHP-2 can also dephosphorylate RasGAP-binding sites on RTKs or Gab1, thereby excluding RasGAP from signaling complexes and promoting Ras activation. Finally, SHP-2 could dephosphorylate the adaptors Cbp/PAG and paxillin on their Csk-binding sites [37]. Because Csk is a negative regulator of Src, this event promotes Src activation. Once activated, Src can affect the Ras/MAPK pathway through incompletely defined mechanisms or via Golgi-bound Ras through a phospholipase C (PLC)γ/Ca2+/RasGRP1 pathway.

The absence of mature thymocytes in these dnSHP-2 mice suggests that SHP-2 plays a role early in T cell development. It is well known that the pre-TCR and TCR complexes play critical roles in T cell development [38]. The protein tyrosine phosphatase SHP-1, which is expressed mainly in hemopoietic cells and is a negative regulator in various signaling pathways, shares a similar structure with SHP-2 in that both have two SH2 domains and a phosphatase domain, although they have relatively low sequence homology [3]. SHP-1 is known to play a role in establishing TCR signaling thresholds that regulate T cell development [39]. It has also been shown to affect both positive and negative selection [40]. Therefore, because SHP-2 is also involved in TCR signaling, it is conceivable that SHP-2 can have regulatory effects on T cell development, especially since we did not observe peripheral T cells in our aggregation attempts.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 81370609).

Disclosure of conflict of interest

None.

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