11 research outputs found
SAP-silencing down-modulates IL-2 and IL-4 gene expression in Jurkat cells.
<p><i>A and B</i>, The Jurkat parental cell (WT) and two clones (ΔSAP-1 and ΔSAP-2), stably expressing two different SAP specific Sh-RNAs (Sh-A3 and Sh-A5 respectively), were stimulated by CD3/CD28 for 60 min for IL-2 (A) or 30 min for IL-4 (B). Quantitative RT-PCR was performed for IL-2 (A) and IL-4 (B) gene expression. Results were normalized to the ribosomal protein, Rpl38, gene expression. Data shown are representative of two independent experiments. <i>C</i>, Whole cell lysates of the Jurkat parental cell (WT) and the two clones ΔSAP-1 and ΔSAP-2 were resolved by SDS-PAGE and SAP extinction was controlled by an anti-SAP immunoblotting. Equal amount of proteins in each lane were assessed by an actin-immunoblotting. Each band was numerically quantitated (indicated in arbitrary units, a.u.) and the SAP/Actin ratio for each cell population is graphically presented (normalized to 100% in Jurkat WT).</p
SAP acts in the CD3-mediated activation of Erk, Akt and PLCγ1 pathways.
<p><i>A</i>, The parental H9 cell (WT) and H9 cells, stably expressing a SAP specific Sh-RNA (ΔSAP), were stimulated with UCHT-1 for the indicated times. 25 µg of proteins were separated by SDS-PAGE and Zap70, Erk and Akt activations were revealed by Western blotting using specific phospho-antibodies. The total amount of each protein in the lysate was shown for comparison. SAP extinction was controlled by an anti-SAP immunoblotting and equal quantities of proteins in each lane were assessed by an anti-Actin immunoblotting. <i>B</i>, parental Jurkat cells (WT) and Jurkat clones expressing either SAP-targeting or CD3ζ-targeting Sh-RNAs (ΔSAP and Δζ respectively) were stimulated or not with UCHT-1 for 10 minutes and lysed. Proteins were immunoprecipited using a phospho-tyrosine antibody (4G10), separated on SDS-PAGE and transferred. PLC-γ1, Slp76 and Grb2 were immunoblotted using specific antibodies. This figure is representative of at least eight experiments in different clones and cell lines.</p
The Adaptor Protein SAP Directly Associates with CD3ζ Chain and Regulates T Cell Receptor Signaling
<div><p>Mutations altering the gene encoding the SLAM associated protein (SAP) are responsible for the X-linked lymphoproliferative disease or XLP1. Its absence is correlated with a defective NKT cells development, a decrease in B cell functions and a reduced T cells and NK cells cytotoxic activities, thus leading to an immunodeficiency syndrome. SAP is a small 128 amino-acid long protein that is almost exclusively composed of an SH2 domain. It has been shown to interact with the CD150/SLAM family of receptors, and in a non-canonical manner with SH3 containing proteins such as Fyn, βPIX, PKCθ and Nck1. It would thus play the role of a minimal adaptor protein. It has been shown that SAP plays an important function in the activation of T cells through its interaction with the SLAM family of receptors. Therefore SAP defective T cells display a reduced activation of signaling events downstream of the TCR-CD3 complex triggering. In the present work, we evidence that SAP is a direct interactor of the CD3ζ chain. This direct interaction occurs through the first ITAM of CD3ζ, proximal to the membrane. Additionally, we show that, in the context of the TCR-CD3 signaling, an Sh-RNA mediated silencing of SAP is responsible for a decrease of several canonical T cell signaling pathways including Erk, Akt and PLCγ1 and to a reduced induction of IL-2 and IL-4 mRNA. Altogether, we show that SAP plays a central function in the T cell activation processes through a direct association with the CD3 complex.</p> </div
SAP associates with NTB-A and CD3ζ in activated T lymphocytes.
<p><i>A</i>, <i>upper-left panel:</i> Jurkat cells were stimulated or not with the anti-CD3ε mAb UCHT1 for 10 minutes, lysed, precleared and GST or GST-SAP pull down experiments were performed. <i>Upper-Middle panel:</i> Jurkat cells were treated with 25 µM of pervanadate for 30 minutes, lysed, precleared and GST-SAP pull-down was performed as above. <i>Upper-Right panel:</i> T-cell blasts were stimulated with UCHT1 for 10 minutes, lysed, precleared and GST-SAP was incubated with cell lysate. Pull downs were immunoblotted with anti-phosphotyrosine (4G10) antibody. <i>B</i>, Jurkat cells were either untreated or treated with 25 µM of pervanadate for 30 minutes, lysed, precleared and GST or GST-SAP were incubated with cell lysates. GST pull downs were immunoblotted for NTB-A or CD3ζ, as indicated. <i>C</i>, T-cell blasts were stimulated or not with UCHT1 for 10 minutes, lysed, precleared and GST or GST-SAP pull downs were performed and immunoblotted for CD3ζ. These experiments were repeated at least 3 times. The position of NTB-A is indicated by an arrow.</p
SAP directly associates with phospho-CD3ζ.
<p><i>A</i>, Jurkat cells were untreated or treated with 25 µM of pervanadate for 30 minutes, lysed and immunoprecipited with an anti-CD3ζ antibody. The immunoprecipitates were resolved by SDS-PAGE and transferred, the membranes were subjected to Far-western blotting with GST alone (<i>panel FW GST</i>) then GST-SAP fusion protein (<i>panel FW GST-SAP</i>), stripped and reprobed with an anti-phosphotyrosine (4G10) antibody (<i>upper panel</i>) and then with an anti-CD3ζ antibody (<i>lower panel</i>). <i>B</i>, Jurkat cells were untreated or treated with 25 µM of pervanadate for 30 minutes, lysed, immunoprecipited for CD3ζ and then immunoblotted for SAP (upper panel). The equal amount of precipitated CD3ζ was assessed by an anti CD3ζ blot (lower panel). These experiments were repeated at least 3 times.</p
SAP partners are exclusively in the membrane fractions.
<p>Jurkat cells were either untreated or treated with 25 µM of pervanadate for 30 minutes. Cells were fractionated by Dounce homogenization and membranes were purified by ultra centrifugation, resolubilized in lysis buffer, precleared and GST or GST-SAP were incubated with the cytosol or membrane fractions. A, pull downs were separated and immunoblotted with anti-phosphotyrosine antibody (4G10). B, crude membrane or cytosolic fractions from 5×10<sup>5</sup> cells were separated and immunoblotted for SAP. Each band was numerically quantitated (indicated in arbitrary units, a.u.) and the percentages indicate the evolution of the band intensity in the stimulated lysate reported to the non-stimulated lysate in each fraction. These experiments were repeated at least four times. TME: Total Membrane Extract, TCE: Total Cytosolic Extract.</p
SAP associates with the first CD3ζ-ITAM motif.
<p><i>A</i>, Schematic view of the generated CD3ζ mutants. <i>B</i>, HeLa cells were transfected with either an empty vector, or with myc-CD3ζ-WT or with mutant myc-CD3ζ as indicated. Forty-eight hours post-transfection the cells were left untreated or were treated with pervanadate for 10 minutes, lysed, precleared and GST-SAP was incubated with the cell lysates. GST-SAP pull downs were immunoblotted with an anti-Myc (9E10) antibody (<i>upper panel</i>) and lysates were immunoblotted with anti-CD3ζ and anti-Ptyr antibodies (middle and lower panels, respectively). <i>C</i>, HeLa cells were transfected with either an empty vector, or with myc-CD3ζ-WT or with myc-CD3ζ- bearing mutations in the first ITAM, and treated as in <i>B</i>. This figure is representative of at least three independent experiments.</p
SHP2 regulates the phosphorylation and internalization of CDCP1.
<p><b>A.</b> HeLa cells stably transfected with an empty vector or with a WT-CDCP1 construct were stably transfected with a SHP2-targeting shRNA (D1 or D2), as indicated. Total cell lysates were prepared and analyzed by western blotting with the antibodies indicated. <b>B.</b> Stable HeLa-CDCP1 and HeLa-CDCP1-shSHP2 D1 cell lines (described above and in the experimental procedures) were first incubated with an anti-CDCP1 antibody at 4°C. The cells were washed and incubated at 37°C for the times indicated, to allow internalization of the CDCP1-antibody complexes. The cells were then incubated again at 4°C with the appropriate secondary antibody, and the amount of CDCP1 remaining at the cell surface was analyzed by flow cytometry. The results are indicated as a percentage of membrane CDCP1 ± SEM for three independent experiments. ns: p > 0.05 *: p = 0.03; ****: p = 10<sup>–4</sup> in non-parametric Student's <i>t</i> tests. The data shown are representative of at least three independent experiments performed in triplicate.</p
Phosphorylated CDCP1 is efficiently pulled down by recombinant SHP2 substrate trapping mutant or SHP2-SH2 domains.
<p>HeLa cells stably expressing CDCP1 were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO<sub>3</sub>), as indicated. Cell lysates were subjected to GST-pull down assays with 5 μg of GST protein alone (GST only), GST fused to a SHP2 substrate trapping mutant (GST-DACS) or GST fused to the SHP2-SH2 domains (GST-SH2), as mentioned in A and B. The affinity-purified complexes were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. In some conditions, particularly in HeLa cells, CDCP1 was detected as two species, the more slowly migrating species being tyrosine-phosphorylated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123472#pone.0123472.g004" target="_blank">Fig 4A and 4B</a>, compare lower and middle panels, the arrowhead indicates the slower migrating species). The data shown are representative of more than eight independent experiments.</p
Primary sequence of intracellular CDCP1.
<p><b>A.</b> Amino acids are numbered from the first intracellular residue. Phosphorylatable tyrosine residues are shown in bold typeface and are numbered. The ITAM-like motif is shown in a box. <b>B.</b> Alignment of the ITAM-like motif of CDCP1 with the consensus sequence of an ITAM motif. Phosphorylatable residues are shown in bold typeface and are numbered according to the corresponding CDCP1 sequence.</p