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. 2005 Jul 6;24(13):2331-41.
doi: 10.1038/sj.emboj.7600706. Epub 2005 Jun 2.

Receptor-stimulated oxidation of SHP-2 promotes T-cell adhesion through SLP-76-ADAP

Jaeyul Kwon  1 , Cheng-Kui Qu, Jin-Soo Maeng, Rustom Falahati, Chunghee Lee, Mark S Williams
Affiliations

Receptor-stimulated oxidation of SHP-2 promotes T-cell adhesion through SLP-76-ADAP

Jaeyul Kwon et al. EMBO J. .

Abstract

Receptor-stimulated generation of intracellular reactive oxygen species (ROS) modulates signal transduction, although the mechanism(s) is unclear. One potential basis is the reversible oxidation of the active site cysteine of protein tyrosine phosphatases (PTPs). Here, we show that activation of the antigen receptor of T cells (TCR), which induces production of ROS, induces transient inactivation of the SH2 domain-containing PTP, SHP-2, but not the homologous SHP-1. SHP-2 is recruited to the LAT-Gads-SLP-76 complex and directly regulates the phosphorylation of key signaling proteins Vav1 and ADAP. Furthermore, the association of ADAP with the adapter SLP-76 is regulated by SHP-2 in a redox-dependent manner. The data indicate that TCR-mediated ROS generation leads to SHP-2 oxidation, which promotes T-cell adhesion through effects on an SLP-76-dependent signaling pathway to integrin activation.

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Figures

Figure 1
Figure 1
TCR stimulation induces oxidative modification of SHP-2. (A) Reduced thiols (PTP-S−) are labeled with PEO-iodoacetyl biotin, but oxidized thiols (PTP-S-OH; sulfenic acid) are not. (B, C) After anti-CD3 stimulation for the indicated times, Jurkat T cells were lysed and labeled with PEO-iodoacetyl biotin as described in Materials and methods. SHP-2 (B) or SHP-1 (C) was immunoprecipitated and biotinylation detected with HRP-conjugated streptavidin and ECL. Equal application of protein was confirmed by immunoblot analysis. The level of biotin incorporation (by densitometry) was corrected for total protein levels and the signal at time=0 was normalized to 100%. The graphs represent the average of three separate experiments±s.e.m. **Significantly different from unstimulated controls; P<0.01. (D) Mouse T-cell blasts were stimulated for the indicated times by anti-CD3 crosslinking. SHP-2 oxidation was measured as in (B). (E) Jurkat T cells were transiently transfected with an expression vector encoding (His)6-SHP-2(C/S) and cells were left unstimulated. Incorporation of biotin into endogenous or tagged SHP-2 was measured as in (B).
Figure 2
Figure 2
SHP-2 oxidation measured by ‘positive labeling'. Human T blasts (A, B) or Jurkat T cells (C, D) were stimulated by anti-CD3 crosslinking for the indicated times. Oxidized thiols were labeled with PEO-iodoacetyl biotin in a ‘positive' labeling method as described in Materials and methods. Biotinylated proteins were isolated on Neutravidin-Sepharose, separated by SDS–PAGE and immunoblotted for SHP-2 (A, C) or SHP-1 (B, D). An aliquot of the lysates was reserved to blot for total SHP-2 or SHP-1. (E, F) Antioxidants inhibit SHP-2 oxidation. (E) Mouse T-cell blasts were preincubated in the absence (−) or presence (+) of 20 mM NAC for 60 min. (F) Jurkat T cells were transfected with an empty vector (−) or one encoding Prx II (+) and prepared as described in Materials and methods. Cells were stimulated by anti-CD3 crosslinking for the indicated times and SHP-2 oxidation was detected as in Figure 1. The level of biotin incorporation (by densitometry) was corrected for total protein levels and the signal at time=0 was normalized to 100%. The graphs represent the average of three separate experiments±s.e.m. **Significantly different from unstimulated controls; P<0.01.
Figure 3
Figure 3
Association of proteins with SHP-2(C/S) upon TCR stimulation. (A) Jurkat T cells were transiently transfected with an empty vector (Vector) or an expression vector encoding HA-SHP-2(C/S) or HA-SHP-2 WT. Cells were stimulated by anti-CD3 crosslinking for 5 min and lysed. Anti-HA immunoprecipitates were blotted with anti-phosphotyrosine and the membrane was stripped and reprobed with antibodies to the indicated proteins. (B–F) Effect of SHP-2(C/S) expression on association of signaling molecules with LAT and Grb2. Jurkat T cells were transfected and stimulated as in (A). (C) Levels of SHP-2 in total cell lysates were determined by immunoblot analysis. (B, D) Anti-LAT or (E, F) anti-Grb2 immunoprecipitates were blotted with (B, E) anti-phosphotyrosine and (D, F) the membranes were stripped and reprobed for the indicated proteins.
Figure 4
Figure 4
Effect of SHP-2(C/S) on phosphorylation and association of proteins with Gads and SLP-76. Jurkat T cells were transiently transfected with an empty vector (Vector) or an expression vector encoding SHP-2(C/S) or SHP-2 WT. Cells were stimulated for 5 min by anti-CD3 crosslinking. Lysates were immunoprecipitated with (A) anti-Gads, (B) anti-SLP-76, (C) anti-Vav1 or (D) anti-ADAP antibody. Immunoprecipitates were separated by SDS–PAGE, immunoblotted for phosphotyrosine and then stripped and reprobed for the indicated proteins. (C) Anti-Vav1 immunoprecipitates were also blotted with a phosphospecific antibody to Vav1 phosphorylated at Y174.
Figure 5
Figure 5
Effect of Prx II on association of proteins with Gads and SLP-76. Jurkat T cells were transfected with an empty vector or an expression vector encoding Prx II, and cells were stimulated by anti-CD3 crosslinking. Lysates were immunoprecipitated with (A) anti-Gads, (B) anti-SLP-76 or (C) anti-ADAP antibody. Immunoprecipitates were separated by SDS–PAGE, immunoblotted for phosphotyrosine and were stripped and reprobed for the indicated proteins. (A) Total cell lysates were probed for Prx II to measure overexpression and β-tubulin to control for loading. (D) Effects of coexpression of SHP-2(C/S) and Prx II. Jurkat T cells were transfected with an empty vector or an expression vector encoding SHP-2(C/S) alone or in combination with one encoding Prx II (SHP-2(C/S)+Prx II) and the cells were stimulated by anti-CD3 crosslinking. Lysates were immunoprecipitated with anti-SLP-76 antibody and probed with antibodies to the indicated proteins. Total lysates were probed for SHP-2 and Prx II to measure expression levels and β-tubulin was probed to control loading.
Figure 6
Figure 6
Effect of SHP-2 or Prx II on TCR-stimulated adhesion to fibronectin and LFA-1 clustering. (A) Jurkat T cells were transiently transfected with a β-gal expression plasmid in the presence of an empty vector (Vector) or an expression vector encoding SHP-2(C/S), SHP-2 WT or Prx II. Adhesion of anti-CD3-stimulated cells to fibronectin-coated wells was performed as described in Materials and methods. The data are expressed as the percent anti-CD3-induced increase in β-gal activity in the wells±s.e.m. and represent the average of triplicate samples in at least six separate experiments. (B, C) Jurkat T cells were transfected with an expression vector for GFP with a two-fold excess of empty vector, or expression vectors for SHP-2(C/S) or Prx II. Cells were stimulated for 15 min by anti-CD3 crosslinking and were stained with anti-human LFA-1 (TS2/4) as in Materials and methods and analyzed by fluorescence microscopy. (B) Representative fields showing LFA-1 staining. Arrows indicate the productively transfected GFP+ cells. (C) The data show the percentage of GFP+ T cells with LFA-1 clustering±s.e.m. from at least five separate experiments with at least 50 GFP+ cells counted per sample. For both experiments, *P<0.05 as compared to vector control cells and **P<0.01 as compared to vector control cells.
Figure 7
Figure 7
Model showing protein associations in the Gads–SLP-76 complex and the proposed targets for SHP-2. (Top) TCR stimulation induces production of ROS (H2O2), which oxidizes SHP-2 present in the Gads–SLP-76 complex. SHP-2 association is independent of SLP-76 and potentially is recruited by another Gads protein bound to the SHP-2 binding adapter Gab2. Oxidation of SHP-2 promotes phosphorylation of ADAP and Vav1, which is translated to increased LFA-1 clustering and T-cell adhesion. (Bottom) In the presence of antioxidants, for example, the effects of ROS are blunted and SHP-2 in the complex remains more active. This leads to less ADAP–SLP-76 association, inhibits LFA-1 clustering and decreases adhesion.
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