Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury.

Neutrophils trigger inflammation-induced acute kidney injury (AKI), a frequent and potentially lethal occurrence in humans. Molecular mechanisms underlying neutrophil recruitment to sites of inflammation have proved elusive. In this study, we demonstrate that SLP-76 (SH2 domain-containing leukocyte phosphoprotein of 76 kD) and ADAP (adhesion and degranulation promoting adaptor protein) are involved in E-selectin-mediated integrin activation and slow leukocyte rolling, which promotes ischemia-reperfusion-induced AKI in mice. By using genetically engineered mice and transduced Slp76(-/-) primary leukocytes, we demonstrate that ADAP as well as two N-terminal-located tyrosines and the SH2 domain of SLP-76 are required for downstream signaling and slow leukocyte rolling. The Tec family kinase Bruton tyrosine kinase is downstream of SLP-76 and, together with ADAP, regulates PI3Kγ (phosphoinositide 3-kinase-γ)- and PLCγ2 (phospholipase Cγ2)-dependent pathways. Blocking both pathways completely abolishes integrin affinity and avidity regulation. Thus, SLP-76 and ADAP are involved in E-selectin-mediated integrin activation and neutrophil recruitment to inflamed kidneys, which may underlie the development of life-threatening ischemia-reperfusion-induced AKI in humans

Studies on ITK-SYK signaling pathways

Chromosomal alterations are frequent causes of cancer. Until now, SYK is reported in two different chromosomal translocation events generating the ITK-SYK-fusion protein in a subset of peripheral T cell lymphomas and the TEL-SYK fusion protein in a case of myelodysplastic syndrome. T lymphocyte-expressed ITK is the only member of the TEC-family of tyrosine kinases reported as a fusion partner in transforming translocations and here we have studied this fusion. The comparison of ITK-SYK with SYK revealed that related tyrosines of ITK-SYK are phosphorylated at the linker- region and at the activation-loop and that the fusion protein localizes to the plasma membrane and potently phosphorylates the adapter proteins SLP-76 and BLNK. Moreover, membrane localization and phosphorylation of adapter substrates are blocked with PI3K inhibitors. SYK, on the other hand, showed phosphorylation at the linker-region, but not at the activation-loop tyrosines and failed to phosphorylate SLP- 76 or BLNK under the same conditions. Since BTK is the predominantly expressed TEC family kinase in B lymphocytes, we engineered the corresponding fusion kinase, BTK-SYK. We then investigated the role of the N-terminal region in the regulation of fusion kinases ITK-SYK, BTK-SYK and TEL-SYK. Unlike ITK-SYK, BTK-SYK showed more nuclear and cytoplasmic localization and PI3K inhibitors, unexpectedly, did not block its capacity to phosphorylate the adapter substrate SLP-76. Interestingly, non-membrane-tethering PH-TH domain-mutants ITK-SYK-R29C and BTK-SYK-R28C potently phosphorylated SLP-76. On the same ground, a TEL-SYK mutant, lacking the dimerization domain, was equally phosphorylated as the full-length fusion protein, but induced highly compromised CD69 upregulation compared with TEL-SYK or ITK- SYK. Further investigations revealed that ITK-SYK-mediated activation of T cells was dependent on the adapter function of SYK-family kinases (SYK or ZAP-70), but independent of their kinase activity. Moreover, SLP-76 adapter function was not only indispensible for ITK-SYK-mediated CD69 upregulation and IL-2 secretion, but also for the phosphorylation of activation-loop tyrosines of SYK. Mutagenesis revealed a hierarchical phosphorylation pattern in the activation of ITK-SYK. In spite of loss of phosphorylation of the tyrosines, known to act as targets in SYK, the fusion protein potently retained phosphorylation capacity for substrate adapter proteins. Phosphorylation-independent constitutive activation was further confirmed by ITK- SYK expression in SYF cells (cells lacking SRC-family kinases), since there was no detectable phosphorylation on target tyrosines, yet the substrate SLP-76 was potently phosphorylated. Altogether, our studies indicate that lack of auto-inhibition renders fusion kinase constitutive activation suggesting that many of the tyrosine phosphorylations known to be critical in the activation of SYK are dispensable for ITK- SYK activation

Immune adaptor ADAP in T cells regulates HIV-1 transcription and cell-cell viral spread via different co-receptors

Background: Immune cell adaptor protein ADAP (adhesion and degranulation-promoting adaptor protein) mediates aspects of T-cell adhesion and proliferation. Despite this, a connection between ADAP and infection by the HIV-1 (human immunodeficiency virus-1) has not been explored. Results: In this paper, we show for the first time that ADAP and its binding to SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) regulate HIV-1 infection via two distinct mechanisms and co-receptors. siRNA down-regulation of ADAP, or expression of a mutant that is defective in associating to its binding partner SLP-76 (termed M12), inhibited the propagation of HIV-1 in T-cell lines and primary human T-cells. In one step, ADAP and its binding to SLP-76 were needed for the activation of NF-κB and its transcription of the HIV-1 long terminal repeat (LTR) in cooperation with ligation of co-receptor CD28, but not LFA-1. In a second step, the ADAP-SLP-76 module cooperated with LFA-1 to regulate conjugate formation between T-cells and dendritic cells or other T-cells as well as the development of the virological synapse (VS) and viral spread between immune cells. Conclusions: These findings indicate that ADAP regulates two steps of HIV-1 infection cooperatively with two distinct receptors, and as such, serves as a new potential target in the blockade of HIV-1 infection

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. © 2015 Hem et al

Differential roles for the adapters Gads and LAT in platelet activation by GPVI and CLEC-2

Background: The adapter proteins SLP-76 and LAT have been shown to play critical roles in the activation of PLCc2 in platelets downstream of GPVI/FcRc and the C-type lectin receptor CLEC-2. SLP-76 is constitutively associated with the adapter Gads in platelets, which also binds to tyrosine phosphorylated LAT, thereby providing a potential pathway of regulation of SLP-76. Objective: In the present study, we have compared the role of Gads alongside that of LAT following activation of the major platelet glycoprotein receptors using mice deficient in the two adapter proteins. Results: Gads was found to be required for the efficient onset of aggregation and secretion in response to submaximal stimulation of GPVI and CLEC-2, but to be dispensable for activation following stronger stimulation of the two receptors. Gads was also dispensable for spreading induced through integrin aIIbb3 or the GPIb–IX–V complex.Further, Gads plays a negligible role in aggregate formation on collagen at an arteriolar rate of shear. In stark contrast, platelets deficient in the adapter LAT exhibit a marked decrease in aggregation and secretion following activation of GPVI and CLEC-2, and are unable to form stable aggregates on collagen at arteriolar shear. Conclusions: The results demonstrate that Gads plays a key role in linking the adapter LAT to SLP-76 in response to weak activation of GPVI and CLEC-2 whereas LAT is required for full activation over a wider range of agonist concentrations. These results reveal the presence of a Gads-independent pathway of platelet activation downstream of LAT

Nuclear pore complex-mediated modulation of TCR signaling is required for naïve CD4+ T cell homeostasis.

Nuclear pore complexes (NPCs) are channels connecting the nucleus with the cytoplasm. We report that loss of the tissue-specific NPC component Nup210 causes a severe deficit of naïve CD4+ T cells. Nup210-deficient CD4+ T lymphocytes develop normally but fail to survive in the periphery. The decreased survival results from both an impaired ability to transmit tonic T cell receptor (TCR) signals and increased levels of Fas, which sensitize Nup210-/- naïve CD4+ T cells to Fas-mediated cell death. Mechanistically, Nup210 regulates these processes by modulating the expression of Cav2 (encoding Caveolin-2) and Jun at the nuclear periphery. Whereas the TCR-dependent and CD4+ T cell-specific upregulation of Cav2 is critical for proximal TCR signaling, cJun expression is required for STAT3-dependent repression of Fas. Our results uncover an unexpected role for Nup210 as a cell-intrinsic regulator of TCR signaling and T cell homeostasis and expose NPCs as key players in the adaptive immune system

Anti-CD20 Therapy Acts via FcγRIIIA to Diminish Responsiveness of Human Natural Killer Cells

Natural killer (NK) immune cells mediate antibody-dependent cellular cytotoxicity (ADCC) by aggregating FcγRIIIA/CD16, contributing significantly to the therapeutic effect of CD20 monoclonal antibodies (mAb). In this study, we show that CD16 ligation on primary human NK cells by the anti-CD20 mAb rituximab or ofatumumab stably impairs the spontaneous cytotoxic response attributable to cross-tolerance of several unrelated NK-activating receptors (including NKG2D, DNAM-1, NKp46, and 2B4). Similar effects were obtained from NK cells isolated from patients with chronic lymphocytic leukemia in an autologous setting. NK cells rendered hyporesponsive in this manner were deficient in the ability of these cross-tolerized receptors to phosphorylate effector signaling molecules critical for NK cytotoxicity, including SLP-76, PLCγ2, and Vav1. These effects were associated with long-lasting recruitment of the tyrosine phosphatase SHP-1 to the CD16 receptor complex. Notably, pharmacologic inhibition of SHP-1 with sodium stibogluconate counteracted CD20 mAb-induced NK hyporesponsiveness, unveiling an unrecognized role for CD16 as a bifunctional receptor capable of engendering long-lasting NK cell inhibitory signals. Our work defines a novel mechanism of immune exhaustion induced by CD20 mAb in human NK cells, with potentially negative implications in CD20 mAb-treated patients where NK cells are partly responsible for clinical efficacy. Cancer Res; 75(19); 1-12. ©2015 AACR

Recruitment of Slp-76 to the Membrane and Glycolipid-Enriched Membrane Microdomains Replaces the Requirement for Linker for Activation of T Cells in T Cell Receptor Signaling

Two hematopoietic-specific adapters, src homology 2 domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224–244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cγ1 phosphorylation, extracellular signal–regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224–244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane

Clnk plays a role in TNF-alpha-induced cell death in murine fibrosarcoma cell line L929

Clnk/MIST在一系列细胞因子依赖的造血细胞系中表达，同时也在一些细胞因子非依赖的肥大细胞系中表达，并且能够与PLC，Grb2，ADAP和HPK-1等蛋白相互作用。 作为Blnk/SLP-76接头分子蛋白家族的第三个成员，Clnk参与免疫受体信号通路的正向调控。但是，在Clnk基因敲除鼠中，Clnk对T细胞，NK细胞和肥大细胞的正常分化和发育并不是必须的。 本论文发现了Clnk在免疫信号通路之外的一些功能。我们发现Clnk在一系列细胞因子非依赖的非造血细胞系中表达，比如L929，B16F10，MCF7和HeLa细胞。在L929细胞中，IL-2和TNF的刺激并不能显著增加...Clnk/MIST is expressed in a variety of cytokine-dependent hematopoietic cell lines as well as some cytokine-independent mast cell lines, and can interact with PLC, Grb2, ADAP and HPK-1. As a third member of the Blnk/SLP-76 adapter family, Clnk is involved in the positive regulation of immunoreceptor signaling. However, Clnk is not essential for normal differentiation and function of T ce...学位：理学硕士院系专业：生命科学学院_生物化学与分子生物学学号：2162012115235

A non-conserved amino acid variant regulates differential signalling between human and mouse CD28

CD28 superagonistic antibodies (CD28SAb) can preferentially activate and expand immunosuppressive regulatory T cells (Treg) in mice. However, pre-clinical trials assessing CD28SAbs for the therapy of autoimmune diseases reveal severe systemic inflammatory response syndrome in humans, thereby implying the existence of distinct signalling abilities between human and mouse CD28. Here, we show that a single amino acid variant within the C-terminal proline-rich motif of human and mouse CD28 (P212 in human vs. A210 in mouse) regulates CD28-induced NF-κB activation and pro-inflammatory cytokine gene expression. Moreover, this Y209APP212 sequence in humans is crucial for the association of CD28 with the Nck adaptor protein for actin cytoskeleton reorganisation events necessary for CD28 autonomous signalling. This study thus unveils different outcomes between human and mouse CD28 signalling to underscore the importance of species difference when transferring results from preclinical models to the bedside