original article t cell activation is reduced by the catalytically … · 2018. 8. 31. ·...

Int J Clin Exp Med 2015;8(4):6568-6577www.ijcem.com /ISSN:1940-5901/IJCEM0006683

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

Baoxia Dong1*, Yubo Gao2*, Xuan Zheng1, Guangxun Gao1, Hongtao Gu1, Xiequn Chen1, Jinyi Zhang3

1Department of Hematology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shanxi Province, China; 2Department of Urology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China; 3Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Ontario, Canada. *Equal contributors.

Received February 3, 2015; Accepted April 3, 2015; Epub April 15, 2015; Published April 30, 2015

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, trans-genic mice

Introduction

SHP-2 is a ubiquitously expressed 70-72-kDa cytosolic tyrosine phosphatase that is implicat-ed 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 pro-tein 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 mole-cules, such as insulin receptor substrate-1 (IRS-1) and Grb2, transmembrane proteins, such as SH2 domain-containing protein tyro-sine phosphatase substrate 1 (SHPS-1), and adhesion molecules such as platelet/endothe-lial 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 resi-dues 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 some-what unclear, although it was found to be required for insulin-induced JNK activation [18].

http://www.ijcem.com

Inactive SHP-2 reduces T cell activation

6569 Int J Clin Exp Med 2015;8(4):6568-6577

(PCR). The studies described below were per-formed in mice that were backcrossed over three generations to the C57BL/6 background.

Flow cytometry

Flow cytometry was performed using a FACScalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Antibodies to the fol-lowing cell surface markers were used (all BD Pharmingen/BD Biosciences, unless stated otherwise): CD3f (145-2C11; in-house hybrid-oma), 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-), phycoery-thrin (PE-), or allophyocyanin-conjugated sec-ondary 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 cul-tured 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-ace-tate (PMA) plus ionomycin, or Con A (all above

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 architec-ture [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-gesta-tion [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 overex-press a catalytically inactive form of SHP-2 spe-

cifically 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 vec-tor p29Δ2 (sal-) that contains a CD2 promoter and enhancer (Figure 1). The transgene frag-ment was purified and micro-injected into CD1 zygote pro-nuclei. Initial genotyping to identify the transgene- bear-ing founders was done by Southern blotting. The found-ers were bred on a C57BL/6 background. Subsequent genotyping to identify mice bear-ing the transgene was done by polymerase chain reaction

Figure 1. Generation of dnSHP-2 transgenic mice. Subcloning of a FLAG-tagged, murine SHP-2 cDNA carrying the classic cysteine 459-to-serine mu-tation 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.


6570 Int J Clin Exp Med 2015;8(4):6568-6577

from Sigma, St. Louis, MO, USA). Thymocy- tes 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 radioac-tivity was measured using an automated b scin-tillation counter.

Measurement of intracellular calcium flux

A total of 7 × 106 LN cells from WT or dnSHP-2 transgenic mice were rested in complete medi-um 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 strepta-vidin. Calcium levels were detected by flow cytometric analysis of the Indo-1 violet-blue fluorescence ratio.

MAPK activation assay

Lysates from unstimulated and stimulated thy-mocytes were immunopreciptated with a mix-ture 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 com-ponents, myelin basic protein (MBP) and [g-32P] ATP. After incubation, the reaction was

terminated and the samples were boiled, elec-trophoresed through SDS-PAGE gel, and trans-ferred to nitrocellulose membrane. The phos-phorylated 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 cen-trifugation at 13,000 × g for 10 min and immu-noprecipitated with the appropriate Ab. Immu- noprecipitates were washed three times with lysis buffer, then boiled with sodium dodecyl sulfate (SDS) sample buffer for 10 min, sepa-rated by SDS-PAGE, and transferred to PVDF membranes (Millipore, Billerica, MA, USA), fol-lowed 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

Figure 2. 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.


6571 Int J Clin Exp Med 2015;8(4):6568-6577

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 prolif-eration 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.

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 thymo-cytes 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 com-pared with their WT littermates (Figure 2). The results from fluorescence-activated cell sorting (FACS) analyses showed no significant differ-ence 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, includ-ing CD5, CD44, and CD25 (data not shown).

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

Our data from F3 mice revealed that proliferation mea-sured by [3H] thymidine incor-poration was reduced in both the thymocytes and LN T cells of dnSHP-2 transgenic mice

Figure 3. Effects of dnSHP-2 on antigen receptor-evoked proliferation. Thymocytes and LN T cells of dnSHP-2 trans-genic 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.

Figure 4. 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 in-dicated times. Lysates were immunoprecipitated with ERK1 and ERK2 anti-bodies, 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).


6572 Int J Clin Exp Med 2015;8(4):6568-6577

SHP-2 involvement in various TCR-evoked signaling functions

Antigen receptor-induced MAPK activation is attenuated in dnSHP-2 thymocytes. To under-stand 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 pro-tein 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].

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

As SHP-2 is a cytosolic tyrosine phosphatase implicated in many different signaling path-ways, we attempted to determine its effects on overall tyrosine phosphorylation and calcium mobilization signaling. Figure 5 shows that sim-ilar levels of anti-CD3 and CD28-stimulated overall tyrosine-phosphorylated proteins were detected in transgenic mice and their WT litter-mates (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).

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

Figure 5. 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.


6573 Int J Clin Exp Med 2015;8(4):6568-6577

As mentioned above, SHP-2 knockout is embry-onic 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 func-tioned 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 overex-pression 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

assessed SAPK/JNK activation following stimu-lation with CD3 plus CD28 antibodies for differ-ent time points by western blot. Although a sig-nificant induction of phosphorylated SAPK/JNK was observed at 2, 5, and 10 min in both WT and dnSHP-2 cell lysates, no significant differ-ence between the groups were found at these time points in response to anti-CD3 or anti-CD28 treatment (Figure 6).

Association and tyrosine phosphorylation sta-tus of possible SHP-2-interacting proteins

As we know, SHP-2 forms a multimeric cytosolic complex with many molecules, such as phos-phoinositide 3-kinase (PI3K), zeta-chain-asso-ciated protein kinase 70 (ZAP-70) and TCR-ζ, which may provide a link between SHP-2 and Ras/MAPK pathway activation [11]. To deter-mine protein association and tyrosine phos-

phorylation 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 anti-body. Our data revealed hyper-phosphorylation of p36 and p110 proteins that were asso-ciated with p85 PI3K in dnSHP-2 transgenic mice, but no affect on the hyperphos-phorylation of PI3K itself (Figure 7). These hyperphos-phorylation proteins had the same sizes as linker for acti-vation of T cells (LAT) and Gab2. When immunoprecipi-tation with ZAP-70 or TCR-ζ antibodies were performed followed by immunoblotting with a phosphotyrosine anti-body, there was no change in the tyrsoine phosphorylation of ZAP-70 or TCR-ζ in dnSHP-2 transgenic mice (Figure 8).

Discussion

SHP-2 is known to play a vital role in mammalian develop-ment and signaling pathways.

Figure 6. Effects of dnSHP-2 on SAPK/JNK activation. Thymocytes from dn-SHP-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 im-munoblotting analyses with a phospho-SAPK/JNK antibody (top panel) and a JNK (JNK1 and JNK2) antibody (bottom panel).

Figure 7. 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).


6574 Int J Clin Exp Med 2015;8(4):6568-6577

tion. 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 func-tions 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 sev-eral targets, which are dephosphorylated by the RTK, then act as an adaptor protein for mobilizing Grb2/SOS. SHP-2 can also phos-phorylate several targets, the dephosphoryla-tion of which will promote MAPK activation. These include tyrosine phosphatase non-receptor type substrate-1 (SHPS-1) and major

T cell maturation/proliferation at both the dou-ble-negative (DN) and double-positive (DP) cell stages. The SHP-2-/- transgenic mice exhibited high efficiency and fidelity of SHP-2 gene dele-tion in thymocytes, mediated by the Cre recom-binase, which is consistent with previous reports using this Cre transgenic mouse line [30]. The SHP-2 deletion significantly sup-pressed progression of DN3 thymocytes to the DN4 stage, as evidenced by DN3 cell accumu-lation and decreased DN4 cell numbers. The reduced DN cell number in the S and G2 phas-es 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 prolifera-tion 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 activa-tion 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-men-tioned multimeric complex that also includes SHP-2. Other TCR-evoked signaling functions, including PI3K acti-vation, SAPK/JNK activation, and apoptosis, were not stud-ied 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 inhibi-

Figure 8. 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).


6575 Int J Clin Exp Med 2015;8(4):6568-6577

vault protein (MVP), for which the precise con-nections 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 phosphory-lated, sequesters Grb2/Sos in the cytoplasm [36]. SHP-2 can also dephosphorylate RasGAP-binding sites on RTKs or Gab1, thereby exclud-ing RasGAP from signaling complexes and pro-moting 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 path-ways, shares a similar structure with SHP-2 in that both have two SH2 domains and a phos-phatase domain, although they have relatively low sequence homology [3]. SHP-1 is known to play a role in establishing TCR signaling thresh-olds 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 con-ceivable that SHP-2 can have regulatory effects on T cell development, especially since we did not observe peripheral T cells in our aggrega-tion attempts.

Acknowledgements

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

Disclosure of conflict of interest

None.

Address correspondence to: Dr. Baoxia Dong, Department of Hematology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shanxi

Province, China. Tel: +86-15619000099; Fax: +86-21-64085875; E-mail: [email protected]

References

[1] Freeman RM Jr, Plutzky J and Neel BG. Identifi-cation of a human src homology 2-containing protein-tyrosine-phosphatase: a putative ho-molog of Drosophila corkscrew. Proc Natl Acad Sci U S A 1992; 89: 11239-11243.

[2] Heuberger J, Kosel F, Qi J, Grossmann KS, Ra-jewsky K and Birchmeier W. Shp2/MAPK sig-naling controls goblet/paneth cell fate deci-sions in the intestine. Proc Natl Acad Sci U S A 2014; 111: 3472-3477.

[3] Giri H, Muthuramu I, Dhar M, Rathnakumar K, Ram U and Dixit M. Protein tyrosine phospha-tase SHP2 mediates chronic insulin-induced endothelial inflammation. Arterioscler Thromb Vasc Biol 2012; 32: 1943-1950.

[4] Nakata S, Fujita N, Kitagawa Y, Okamoto R, Ogita H and Takai Y. Regulation of platelet-de-rived growth factor receptor activation by afa-din through SHP-2: implications for cellular morphology. J Biol Chem 2007; 282: 37815-37825.

[5] Xu D and Qu CK. Protein tyrosine phosphatas-es in the JAK/STAT pathway. Front Biosci 2008; 13: 4925-4932.

[6] Klein AL, Berkaw MN, Buse MG and Ball LE. O-linked N-acetylglucosamine modification of insulin receptor substrate-1 occurs in close proximity to multiple SH2 domain binding mo-tifs. Mol Cell Proteomics 2009; 8: 2733-2745.

[7] Ahmed Z, Lin CC, Suen KM, Melo FA, Levitt JA, Suhling K and Ladbury JE. Grb2 controls phos-phorylation of FGFR2 by inhibiting receptor ki-nase and Shp2 phosphatase activity. J Cell Biol 2013; 200: 493-504.

[8] Quintanar-Audelo M, Yusoff P, Sinniah S, Chan-dramouli S and Guy GR. Sprouty-related Ena/vasodilator-stimulated phosphoprotein homol-ogy 1-domain-containing protein (SPRED1), a tyrosine-protein phosphatase non-receptor type 11 (SHP2) substrate in the Ras/extracel-lular signal-regulated kinase (ERK) pathway. J Biol Chem 2011; 286: 23102-23112.

[9] Liu G, Place AT, Chen Z, Brovkovych VM, Vogel SM, Muller WA, Skidgel RA, Malik AB and Min-shall RD. ICAM-1-activated Src and eNOS sig-naling increase endothelial cell surface PE-CAM-1 adhesivity and neutrophil transmi- gration. Blood 2012; 120: 1942-1952.

[10] Dittrich A, Quaiser T, Khouri C, Gortz D, Monni-gmann M and Schaper F. Model-driven experi-mental analysis of the function of SHP-2 in IL-6-induced Jak/STAT signaling. Mol Biosyst 2012; 8: 2119-2134.

[11] Yokosuka T, Takamatsu M, Kobayashi-Imanishi W, Hashimoto-Tane A, Azuma M and Saito T.

mailto:[email protected]


6576 Int J Clin Exp Med 2015;8(4):6568-6577

Programmed cell death 1 forms negative co-stimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phospha-tase SHP2. J Exp Med 2012; 209: 1201-1217.

[12] Maus M, Medgyesi D, Kovesdi D, Csuka D, Koncz G and Sarmay G. Grb2 associated bind-er 2 couples B-cell receptor to cell survival. Cell Signal 2009; 21: 220-227.

[13] Feng GS. Shp-2 tyrosine phosphatase: signal-ing one cell or many. Exp Cell Res 1999; 253: 47-54.

[14] Liu X, Zheng H and Qu CK. Protein tyrosine phosphatase Shp2 (Ptpn11) plays an impor-tant role in maintenance of chromosome sta-bility. Cancer Res 2012; 72: 5296-5306.

[15] Mali RS, Ma P, Zeng LF, Martin H, Ramdas B, He Y, Sims E, Nabinger S, Ghosh J, Sharma N, Munugalavadla V, Chatterjee A, Li S, Sandusky G, Craig AW, Bunting KD, Feng GS, Chan RJ, Zhang ZY and Kapur R. Role of SHP2 phospha-tase in KIT-induced transformation: identifica-tion of SHP2 as a druggable target in diseases involving oncogenic KIT. Blood 2012; 120: 2669-2678.

[16] Song KS, Choi JK and Ahn DW. Src homology 2-containing protein tyrosine phosphatase-2 acts as a negative regulator for MUC5AC tran-scription via the inhibition of the ERK1/2 MAPK signalling pathway in the airway. Acta Physiol (Oxf) 2013; 208: 245-250.

[17] Fang X, Lang Y, Wang Y, Mo W, Wei H, Xie J and Yu M. Shp2 activates Fyn and Ras to regulate RBL-2H3 mast cell activation following Fcepsil-onRI aggregation. PLoS One 2012; 7: e40566.

[18] Fukunaga K, Noguchi T, Takeda H, Matozaki T, Hayashi Y, Itoh H and Kasuga M. Requirement for protein-tyrosine phosphatase SHP-2 in in-sulin-induced activation of c-Jun NH(2)-termi-nal kinase. J Biol Chem 2000; 275: 5208-5213.

[19] Amin AR, Thakur VS, Paul RK, Feng GS, Qu CK, Mukhtar H and Agarwal ML. SHP-2 tyrosine phosphatase inhibits p73-dependent apopto-sis and expression of a subset of p53 target genes induced by EGCG. Proc Natl Acad Sci U S A 2007; 104: 5419-5424.

[20] Hartman ZR, Schaller MD and Agazie YM. The tyrosine phosphatase SHP2 regulates focal ad-hesion kinase to promote EGF-induced lamel-lipodia persistence and cell migration. Mol Cancer Res 2013; 11: 651-664.

[21] Lapinski PE, Meyer MF, Feng GS, Kamiya N and King PD. Deletion of SHP-2 in mesenchymal stem cells causes growth retardation, limb and chest deformity, and calvarial defects in mice. Dis Model Mech 2013; 6: 1448-1458.

[22] Lee HH, Lee HC, Chou CC, Hur SS, Osterday K, del Alamo JC, Lasheras JC and Chien S. Shp2 plays a crucial role in cell structural orientation

and force polarity in response to matrix rigidity. Proc Natl Acad Sci U S A 2013; 110: 2840-2845.

[23] Qu CK, Shi ZQ, Shen R, Tsai FY, Orkin SH and Feng GS. A deletion mutation in the SH2-N do-main of Shp-2 severely suppresses hematopoi-etic cell development. Mol Cell Biol 1997; 17: 5499-5507.

[24] Saxton TM, Henkemeyer M, Gasca S, Shen R, Rossi DJ, Shalaby F, Feng GS and Pawson T. Abnormal mesoderm patterning in mouse em-bryos mutant for the SH2 tyrosine phospha-tase Shp-2. EMBO J 1997; 16: 2352-2364.

[25] Takahashi S, Inatome R, Yamamura H and Yanagi S. Isolation and expression of a novel mitochondrial septin that interacts with CRMP/CRAM in the developing neurones. Genes Cells 2003; 8: 81-93.

[26] Lai LA, Zhao C, Zhang EE and Feng GS. The Shp-2 Tyrosine Phosphatase. In: Arino J, Alex-ander D, editors. Protein Phosphatases. Berlin: Springer-Verlag; 2003. pp. 275-299.

[27] Li K, Leung AW, Guo Q, Yang W and Li JY. Shp2-dependent ERK signaling is essential for in-duction of Bergmann glia and foliation of the cerebellum. J Neurosci 2014; 34: 922-931.

[28] Nguyen TV, Ke Y, Zhang EE and Feng GS. Con-ditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals. J Immunol 2006; 177: 5990-5996.

[29] Koelsch U, Schraven B and Simeoni L. SIT and TRIM determine T cell fate in the thymus. J Im-munol 2008; 181: 5930-5939.

[30] Crompton T, Gilmour KC and Owen MJ. The MAP kinase pathway controls differentiation from double-negative to double-positive thy-mocyte. Cell 1996; 86: 243-251.

[31] Chan G, Cheung LS, Yang W, Milyavsky M, Sanders AD, Gu S, Hong WX, Liu AX, Wang X, Barbara M, Sharma T, Gavin J, Kutok JL, Iscove NN, Shannon KM, Dick JE, Neel BG and Braun BS. Essential role for Ptpn11 in survival of he-matopoietic stem and progenitor cells. Blood 2011; 117: 4253-4261.

[32] Schneider H and Rudd CE. Tyrosine phospha-tase SHP-2 binding to CTLA-4: absence of di-rect YVKM/YFIP motif recognition. Biochem Biophys Res Commun 2000; 269: 279-283.

[33] Salmond RJ, Huyer G, Kotsoni A, Clements L and Alexander DR. The src homology 2 do-main-containing tyrosine phosphatase 2 regu-lates primary T-dependent immune responses and Th cell differentiation. J Immunol 2005; 175: 6498-6508.

[34] Lee KM, Chuang E, Griffin M, Khattri R, Hong DK, Zhang W, Straus D, Samelson LE, Thomp-son CB and Bluestone JA. Molecular basis of T cell inactivation by CTLA-4. Science 1998; 282: 2263-2266.


6577 Int J Clin Exp Med 2015;8(4):6568-6577

[35] Nakaseko C, Miyatake S, Iida T, Hara S, Abe R, Ohno H, Saito Y and Saito T. Cytotoxic T lym-phocyte antigen 4 (CTLA-4) engagement deliv-ers an inhibitory signal through the membrane-proximal region in the absence of the tyrosine motif in the cytoplasmic tail. J Exp Med 1999; 190: 765-774.

[36] Hanafusa H, Torii S, Yasunaga T, Matsumoto K and Nishida E. Shp2, an SH2-containing pro-tein-tyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephos-phorylating and inactivating the inhibitor Sprouty. J Biol Chem 2004; 279: 22992-22995.

[37] Dance M, Montagner A, Salles JP, Yart A and Raynal P. The molecular functions of Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway. Cell Signal 2008; 20: 453-459.

[38] Ren Y, Meng S, Mei L, Zhao ZJ, Jove R and Wu J. Roles of Gab1 and SHP2 in paxillin tyrosine dephosphorylation and Src activation in re-sponse to epidermal growth factor. J Biol Chem 2004; 279: 8497-8505.

[39] Hebeisen M, Baitsch L, Presotto D, Baumgaert-ner P, Romero P, Michielin O, Speiser DE and Rufer N. SHP-1 phosphatase activity counter-acts increased T cell receptor affinity. J Clin In-vest 2013; 123: 1044-1056.

[40] Fu G, Casas J, Rigaud S, Rybakin V, Lambolez F, Brzostek J, Hoerter JA, Paster W, Acuto O, Cheroutre H, Sauer K and Gascoigne NR. The-mis sets the signal threshold for positive and negative selection in T-cell development. Na-ture 2013; 504: 441-445.

original article t cell activation is reduced by the catalytically … · 2018. 8. 31. ·...

Documents