Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7

BACKGROUND: The activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd. RESULTS: TSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239–256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239–334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239–334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells. CONCLUSION: These data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239–334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined

T Cell Specific Adapter Protein (TSAd) Interacts with Tec Kinase ITK to Promote CXCL12 Induced Migration of Human and Murine T Cells

The chemokine CXCL12/SDF-1α interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein β subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail. We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd\u27s effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd\u27s ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses. We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements

T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells

Background The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated. Results To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr280 (pTyr280) and pTyr305. These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr280 and pTyr305 on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck. Conclusions TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells

SH2D2A modulates T cell mediated protection to a B cell derived tumor in transgenic mice.

BACKGROUND: T cell specific adapter protein (TSAd), encoded by the SH2D2A gene, modulates signaling downstream of the T cell receptor (TCR). Young, unchallenged SH2D2A-deficient C57BL/6 mice exhibit a relatively normal immune phenotype. To address whether SH2D2A regulates physiologic immune responses, SH2D2A-deficient TCR-transgenic BALB/c mice were generated. The transgenic TCR recognizes a myeloma-derived idiotypic (Id) peptide in the context of the major histocompatibility complex (MHC) class II molecule I-E(d), and confers T cell mediated resistance to transplanted multiple myeloma development in vivo. PRINCIPAL FINDINGS: The immune phenotype of SH2D2A-deficient C57BL/6 and BALB/c mice did not reveal major differences compared to the corresponding wild type mice. When challenged with myeloma cells, Id-specific TCR-transgenic BALB/c mice lacking SH2D2A displayed increased resistance towards tumor development. Tumor free TCR-transgenic SH2D2A-deficient mice had higher numbers of Id-specific single positive CD4+ thymocytes compared to TCR-transgenic wild-type mice. CONCLUSION: Our results suggest a modulatory role for SH2D2A in T cell mediated immune surveillance of cancer. However, it remains to be established whether its effect is T-cell intrinsic. Further studies are required to determine whether targeting SH2D2A function in T cells may be a potential adjuvant in cancer immunotherapy

T Cell Specific Adapter Protein (TSAd) Interacts with Tec Kinase ITK to Promote CXCL12 Induced Migration of Human and Murine T Cells

The chemokine CXCL12/SDF-1α interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein β subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail. We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd's effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd's ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses. We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements.This article is from PLoS ONE 5 (2010): e9761, doi:10.1371/journal.pone.0009761. Posted with permission.</p

TCR transgenic <i>SH2D2A</i> −<b>/</b>− mice are resistant towards transplanted myeloma.

<p>Non-transgenic BALB/c and Id-specific TCR-transgenic (Id-TCR) BALB/c mice with (+/+) or without (−/−) <i>SH2D2A</i> expression were injected subcutaneously with low dose MOPC315 cells (160 000 cells). Tumor development was followed by palpation. (A, B) A tumor-free mouse was defined as a mouse that did not have a palpable tumor during the course of the experiment. The plots display tumor-take for (A) non-transgenic BALB/c mice with (n = 9) or without (n = 11) <i>SH2D2A</i> expression and (B) Id-specific TCR-transgenic BALB/c mice with (n = 23) or without (n = 23) <i>SH2D2A</i> expression. P values were calculated with two-tailed log rank test. Ns = non-significant.(C, D) Splenic CD4+ T cells were isolated from surviving tumor-free mice and stimulated <i>in vitro</i> with Id-positive F9 cells. Cells were labeled with GB113, recognizing the Id-specific transgenic TCR, and anti-CD69, CD44, CD62L or CD25 mAbs, prior to and after 24, 48 and 72 hours in culture with Id-positive cells. (C) FACS plots gated on GB113+ CD4+ T cells from one representative experiment are shown. (D) The diagram show the median value of the MFI (median fluorescent intensity) of CD69 at the indicated time points (n = 11) with SD. P-values were calculated with two-tailed, unpaired student t test, * indicate significant differences (at 48 hours: p = 0,003; at 72 hours: p = 0,03).</p

<i>SH2D2A</i>-deficient mice have increased numbers of Id-specific TCR–transgenic SP CD4+ thymocytes.

<p>The graphs represents from the left; “UC” –unchallenged mice, i.e. control Id-specific TCR-transgenic mice that did not receive tumor cells, wild-type (<i>SH2D2A</i>+/+) and <i>SH2D2A</i> deficient (<i>SH2D2A</i>−/−) mice that had to be sacrificed due to development of large tumors (tumor volume > 1 cm³) (T = tumor) or that was tumor free at the end of the experiment 70 days after tumor inoculation (TF = tumor free). These tumor-free mice never had a palpable tumor during the course of the experiment, or they had a tumor that never reached 1 cm³ and that disappeared prior to the end of the experiment. The median values are shown as lines in the diagrams. (A, B) Thymocytes from mice with indicated genotypes were labeled with anti Id-TCR (GB113), anti-CD4 and anti-CD8 mAbs and their expressions were monitored by flow cytometry. The total number of Id-specific TCR-transgenic (GB113+) double positive (DP, CD4+ CD8+) and single positive CD4+ (SP, CD4+ CD8−) thymocytes is indicated for each genotype. (C) The diagram display the number of GB113+ CD4+ T cells in the tumor-draining lymph node of the same mice as in A and B. Of note is that the number of GB113+ CD4+ T cells in unchallenged mice is very low due to the small size of the draining lymph node in the absence of tumor challenge. (D) The GB113+ CD4+ T cells from the draining lymph node were labeled with anti-CD69 mAb, and their expression was monitored by flow cytometry. The diagram shows the median fluorescence intensity (MFI) of CD69 divided by the MFI of isotype control in GB113+ CD4+ T cells. P values were calculated with two-tailed Mann-Whitney U test. Ns = non-significant.</p