scholarly journals Functional Cooperation between c-Cbl and Src-Like Adaptor Protein 2 in the Negative Regulation of T-Cell Receptor Signaling

2002 ◽  
Vol 22 (12) ◽  
pp. 4241-4255 ◽  
Author(s):  
Michael P. Loreto ◽  
Donna M. Berry ◽  
C. Jane McGlade
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinases ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Sh2 Domain ◽  
Jurkat T Cells ◽  
Activated T Cells ◽  
Amino Terminal ◽  
Carboxy Terminal

ABSTRACT Adaptor proteins assemble multiprotein signaling complexes, enabling the transduction of intracellular signals. While many adaptor proteins positively regulate signaling in this manner, a subgroup of adaptors function as negative regulators. Here we report the identification of a hematopoiesis-specific adaptor protein that we have designated Src-like adaptor protein 2 (SLAP-2). SLAP-2 is most closely related to SLAP and contains a Src homology 3 (SH3) domain and an SH2 domain, as well as an amino-terminal myristoylation site that mediates SLAP-2 association with membranes. Following stimulation of primary thymocytes with anti-CD3 and anti-CD28, SLAP-2 coimmunoprecipitates with tyrosine-phosphorylated c-Cbl and an unidentified protein of approximately 72 kDa. In activated Jurkat T cells, SLAP-2 also binds an additional 70-kDa phosphoprotein, identified as ZAP-70. Binding of SLAP-2 to both p72 and ZAP-70 is dependent on its SH2 domain, while c-Cbl interacts with the carboxy-terminal region. Overexpression of wild-type SLAP-2 alone or in combination with c-Cbl in Jurkat T cells leads to inhibition of T-cell antigen receptor-induced activation of nuclear factor of activated T cells. The inhibitory effect of SLAP-2 requires the carboxy-terminal c-Cbl binding region. Expression of SLAP-2 with SYK or ZAP-70 in COS cells or Jurkat T cells causes the degradation of these kinases, and SLAP-2 overexpression in Jurkat T cells reduces the surface expression of CD3. These results suggest that the mechanism of action of SLAP-2 and the related protein SLAP is to promote c-Cbl-dependent degradation of the tyrosine kinases SYK and ZAP-70 and down-regulation of CD3 at the cell surface.

Inhibition of T cell activation and function by the adaptor protein CIN85

2019 ◽  
Vol 12 (567) ◽  
pp. eaav4373 ◽  
Author(s):  
Mei Suen Kong ◽  
Akiko Hashimoto-Tane ◽  
Yusuke Kawashima ◽  
Machie Sakuma ◽  
Tadashi Yokosuka ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
Tcr Signaling ◽  
Signaling Complex

T cell activation is initiated by signaling molecules downstream of the T cell receptor (TCR) that are organized by adaptor proteins. CIN85 (Cbl-interacting protein of 85 kDa) is one such adaptor protein. Here, we showed that CIN85 limited T cell responses to TCR stimulation. Compared to activated wild-type (WT) T cells, those that lacked CIN85 produced more IL-2 and exhibited greater proliferation. After stimulation of WT T cells with their cognate antigen, CIN85 was recruited to the TCR signaling complex. Early TCR signaling events, such as phosphorylation of ζ-chain–associated protein kinase 70 (Zap70), Src homology 2 (SH2) domain–containing leukocyte protein of 76 kDa (SLP76), and extracellular signal–regulated kinase (Erk), were enhanced in CIN85-deficient T cells. The inhibitory function of CIN85 required the SH3 and PR regions of the adaptor, which associated with the phosphatase suppressor of TCR signaling–2 (Sts-2) after TCR stimulation. Together, our data suggest that CIN85 is recruited to the TCR signaling complex and mediates inhibition of T cell activation through its association with Sts-2.

scholarly journals Src-like Adaptor Protein (Slap) Is a Negative Regulator of T Cell Receptor Signaling

2000 ◽  
Vol 191 (3) ◽  
pp. 463-474 ◽  
Author(s):  
Tomasz Sosinowski ◽  
Akhilesh Pandey ◽  
Vishva M. Dixit ◽  
Arthur Weiss
Keyword(s):  
T Cells ◽  
T Cell ◽  
Interleukin 2 ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Jurkat Cells ◽  
Negative Regulator ◽  
Tcr Signaling ◽  
Activated T Cells ◽  
Sh2 Domains

Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)–, and interleukin 2–dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3ζ, ZAP-70, SH2 domain–containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

scholarly journals SHP2-interacting Transmembrane Adaptor Protein (SIT), A Novel Disulfide-linked Dimer Regulating Human T Cell Activation

1999 ◽  
Vol 189 (8) ◽  
pp. 1181-1194 ◽  
Author(s):  
Anne Marie-Cardine ◽  
Henning Kirchgessner ◽  
Eddy Bruyns ◽  
Andrej Shevchenko ◽  
Matthias Mann ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tyrosine Phosphatase ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Sh2 Domain ◽  
Jurkat Cells ◽  
Activated T Cells ◽  
Human T Cell

T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain–containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes. After tyrosine phosphorylation by src and possibly syk protein tyrosine kinases SIT recruits the SH2 domain–containing tyrosine phosphatase SHP2 via an immunoreceptor tyrosine-based inhibition motif. Overexpression of SIT in Jurkat cells downmodulates T cell receptor– and phytohemagglutinin-mediated activation of the nuclear factor of activated T cells (NF-AT) by interfering with signaling processes that are probably located upstream of activation of phospholipase C. However, binding of SHP2 to SIT is not required for inhibition of NF-AT induction, suggesting that SIT not only regulates NF-AT activity but also controls NF-AT unrelated pathways of T cell activation involving SHP2.

scholarly journals Ribp, a Novel Rlk/Txk- and Itk-Binding Adaptor Protein That Regulates T Cell Activation

1999 ◽  
Vol 190 (11) ◽  
pp. 1657-1668 ◽  
Author(s):  
Keshava Rajagopal ◽  
Connie L. Sommers ◽  
Donna C. Decker ◽  
Elizabeth O. Mitchell ◽  
Ulf Korthauer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Interferon Γ ◽  
Activated T Cells ◽  
Two Hybrid

A novel T cell–specific adaptor protein, RIBP, was identified based on its ability to bind Rlk/Txk in a yeast two-hybrid screen of a mouse T cell lymphoma library. RIBP was also found to interact with a related member of the Tec family of tyrosine kinases, Itk. Expression of RIBP is restricted to T and natural killer cells and is upregulated substantially after T cell activation. RIBP-disrupted knockout mice displayed apparently normal T cell development. However, proliferation of RIBP-deficient T cells in response to T cell receptor (TCR)-mediated activation was significantly impaired. Furthermore, these activated T cells were defective in the production of interleukin (IL)-2 and interferon γ, but not IL-4. These data suggest that RIBP plays an important role in TCR-mediated signal transduction pathways and that its binding to Itk and Rlk/Txk may regulate T cell differentiation.

scholarly journals Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Matthias Kästle ◽  
Camilla Merten ◽  
Roland Hartig ◽  
Thilo Kaehne ◽  
Ardiyanto Liaunardy-Jopeace ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Enzymatic Activity ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Sh2 Domain ◽  
Jurkat T Cells

Abstract Background Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. Methods Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. Results Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. Conclusions Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. Major findings Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.

scholarly journals Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

2019 ◽  
Author(s):  
Elizabeth A. DeRiso ◽  
Andrea L. Szymczak-Workman ◽  
Angela Montecalvo ◽  
Joanne M. Murphy ◽  
Maria-Cristina Seminario ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Tyrosine Kinases ◽  
Adaptor Proteins ◽  
Cell Receptor ◽  
Human T Cells ◽  
T Cell Receptor Signaling ◽  
Signaling Microclusters ◽  
Cell Receptor Signaling

AbstractThe NF-κB essential modulator protein (NEMO) is required for activation of canonical NF-κB by the T cell antigen receptor (TCR). However, the subcellular localization of NEMO during this process is not well understood. By dynamically imaging fluorescent NEMO chimeras in live human T cells, we demonstrate that NEMO is rapidly recruited into TCR microclusters via domains previously implicated in the recognition of linear and K63-linked polyubiquitin. The recruitment of NEMO into TCR microclusters requires the activities of the tyrosine kinases Lck and ZAP-70, but not the adaptor proteins LAT or SLP-76. Thus, our findings reveal that the pathways leading from TCR to NF-κB bifurcate downstream of ZAP-70 to independently control the recruitment and activation of NEMO.

scholarly journals RIAM Interacts with the Hematopoietic-Specific Adaptor Protein Gads and Forms a LAT-Independent Node of Signal Integration That Regulates Activation of PLC-γ1

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4138-4138
Author(s):  
Kankana Bardhan ◽  
Nikolaos Patsoukis ◽  
Donna M Berry ◽  
Jane McGlade ◽  
Vassiliki A. Boussiotis
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Adaptor Protein ◽  
Ph Domain ◽  
Activated T Cells ◽  
Independent Manner ◽  
C Terminus

Abstract TCR stimulation triggers the activation of protein tyrosine kinases resulting in phosphorylation of the adaptor protein LAT. SLP-76, interacts constitutively with PLC-γ1 and with the SH3 domain of Gads, which via its SH2 domain mediates inducible recruitment of SLP-76 and PLC-γ1 to LAT, upon T cell activation. PLC-γ1 hydrolyzes phosphatidylinositol-4, 5 bisphosphate [PI(4,5)P2], generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), second messengers responsible for mediating intracellular calcium release and activation of downstream signals. The adaptor protein RIAM constitutively interacts with PLC-γ1 and is required for PLC-γ1 activation. RIAM is a multidomain protein with a small N-terminus proline-rich region, two coiled-coiled regions, sequential Ras association (RA) and pleckstrin homology (PH) domains, and a large C-terminus proline-rich region, which interacts with PLC-γ1. The RA domain of RIAM has specificity for Rap1-GTP whereas the PH domain binds to the PLC-γ1 substrate PI(4,5)P2. The RA-PH domain region of RIAM functions as a single structural unit and mediates translocation of RIAM to the plasma membrane upon T cell activation. Previously, we determined that RIAM deficiency results in impaired activation of PLC-γ1 in spite of the formation of the PLC-γ1-SLP-76-LAT complex, suggesting perhaps somewhat paradoxically, that PLC-γ1-SLP-76-LAT signalosome is not sufficient to mediate distal signaling in the absence of RIAM. This observation indicated that RIAM mediates its effects at a level distal to SLP-76-LAT or through a signaling pathway parallel but distinct from SLP-76-Gads-LAT. Here we investigated whether RIAM forms a signalosome parallel to PLC-γ1-SLP-76-Gads and whether such pathway might be involved in the activation of PLC-γ1. Using primary T lymphocytes and Jurkat T cells stimulated via TCR/CD3 and CD28 we determined that RIAM constitutively interacted with Gads as determined by immunoprecipitation with RIAM-specific antibody followed by Gads immunoblot. To determine whether the interaction between RIAM and Gads was direct, we employed an in vitro protein association assay. Glutathione S-transferase (GST) and GST-fusion protein of Gads were coupled to glutathione-sepharose and incubated with [35S]methionine-labeled RIAM or luciferase, as negative control. Gads bound to [35S]methionine-labeled RIAM indicating that RIAM interacts directly with Gads. We further examined domain-specific interaction of RIAM with endogenous Gads using GST fusion proteins of RIAM. We determined a constitutive interaction between Gads and GST fusion proteins of full-length RIAM or C-terminus region of RIAM. Although a number of tyrosine phosphorylated proteins were associated with the RIAM-Gads complex upon T cell activation, LAT was not detected among the components of this complex as determined by immunoblot with anti-phosphotyrosine-specific or LAT-specific antibodies. Using a GST fusion protein of the RA-PH domain of RIAM we determined that, surprisingly, Gads displayed activation-dependent interaction with the RA-PH domain, which mediates the recruitment of RIAM to the plasma membrane upon T cell activation. Furthermore, in addition to Gads, SLP-76 and PLC-γ1 were recruited to the RA-PH domain of RIAM in activated T cells. To determine whether RIAM and Gads had a synergistic effect on IL-2 transcription, we performed luciferase-based reporter assays using a reporter construct driven by the entire IL-2 promoter or by NFAT binding sequences. We found that RIAM and Gads had a synergistic effect on IL-2 and on NFAT-mediated transcriptional activation, which depends on PLC-γ1. Thus, via its C-terminus region, RIAM directly and constitutively interacts with Gads. In addition, via its RA-PH domain, RIAM mediates an activation-dependent interaction with Gads and serves as a docking site recruiting the PLC-γ1-SLP-76-Gads complex to the plasma membrane in a LAT-independent manner. These findings indicate a crosstalk between RIAM and SLP-76 in the activation of PLC-γ1 and reveal a previously unidentified, alternative signaling pathway leading to Gads-SLP-76 recruitment to the plasma membrane of activated T cells in a LAT-independent manner. Disclosures No relevant conflicts of interest to declare.

scholarly journals T Cell Costimulation by CD6 Is Dependent on Bivalent Binding of a GADS/SLP-76 Complex

2017 ◽  
Vol 37 (11) ◽  
Author(s):  
Johannes Breuning ◽  
Marion H. Brown
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
T Cell Activation ◽  
Cell Activation ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Cell Surface Receptor ◽  
Surface Receptor ◽  
Biochemical Approach

ABSTRACT The cell surface receptor CD6 regulates T cell activation in both activating and inhibitory manners. The adaptor protein SLP-76 is recruited to the phosphorylated CD6 cytoplasmic Y662 residue during T cell activation, providing an activating signal to T cells. In this study, a biochemical approach identified the SH2 domain-containing adaptor protein GADS as the dominant interaction partner for the CD6 cytoplasmic Y629 residue. Functional experiments in human Jurkat and primary T cells showed that both mutations Y629F and Y662F abolished costimulation by CD6. In addition, a restraint on T cell activation by CD6 was revealed in primary T cells expressing CD6 mutated at Y629 and Y662. These data are consistent with a model in which bivalent recruitment of a GADS/SLP-76 complex is required for costimulation by CD6.

scholarly journals Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

1998 ◽  
Vol 9 (10) ◽  
pp. 2715-2727 ◽  
Author(s):  
Nadine C. Romzek ◽  
Estelle S. Harris ◽  
Cheryl L. Dell ◽  
Jeffrey Skronek ◽  
Elizabeth Hasse ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Β1 Integrin ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Stimulation Of

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in β1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the β1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human β1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the β1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the β1 integrin with the activating β1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the β1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of β1 integrin structure and function in human T cells.

scholarly journals Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1

2018 ◽  
Vol 11 (560) ◽  
pp. eaat3178 ◽  
Author(s):  
Nathan H. Roy ◽  
Joanna L. MacKay ◽  
Tanner F. Robertson ◽  
Daniel A. Hammer ◽  
Janis K. Burkhardt
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
Actin Polymerization ◽  
Adaptor Protein ◽  
Leading Edge ◽  
Adaptor Proteins ◽  
Substrate Stiffness ◽  
Lymphocyte Function ◽  
Molecular Events

T cell entry into inflamed tissue involves firm adhesion, spreading, and migration of the T cells across endothelial barriers. These events depend on “outside-in” signals through which engaged integrins direct cytoskeletal reorganization. We investigated the molecular events that mediate this process and found that T cells from mice lacking expression of the adaptor protein Crk exhibited defects in phenotypes induced by the integrin lymphocyte function–associated antigen 1 (LFA-1), namely, actin polymerization, leading edge formation, and two-dimensional cell migration. Crk protein was an essential mediator of LFA-1 signaling–induced phosphorylation of the E3 ubiquitin ligase c-Cbl and its subsequent interaction with the phosphatidylinositol 3-kinase (PI3K) subunit p85, thus promoting PI3K activity and cytoskeletal remodeling. In addition, we found that Crk proteins were required for T cells to respond to changes in substrate stiffness, as measured by alterations in cell spreading and differential phosphorylation of the force-sensitive protein CasL. These findings identify Crk proteins as key intermediates coupling LFA-1 signals to actin remodeling and provide mechanistic insights into how T cells sense and respond to substrate stiffness.

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