The many faces of nitric oxide: cytotoxic, cytoprotective or both

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74882/1/j.1365-2982.2007.00958.x.pd

Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4β1

Stimulation by chemokines of integrin α4β1-dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4β1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4β1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase-inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4β1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4β1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4β1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76-, ADAP-, and Pyk2-regulated cell adhesion involving α4β1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4β1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation.This work was supported by grants SAF2011-24022 from Ministerio de Economía y Competitividad, RD12/0036/0061, and S2010/BMD-2314 from Comunidad de Madrid to J.T.Peer Reviewe

A leucine zipper in the N terminus confers membrane association to SLP-65

Membrane recruitment of adaptor proteins is crucial for coupling antigen receptors to downstream signaling events. Despite the essential function of the B cell adaptor SLP-65, the mechanism of its recruitment to the plasma membrane is not yet understood. Here we show that a highly conserved leucine zipper in the SLP-65 N terminus is responsible for membrane association. Alterations in the N terminus abolished SLP-65 membrane localization and activity, both of which were restored by replacement of the N terminus with a myristoylation signal. The N terminus is an autonomous domain that confers specific localization and function when transferred to green fluorescent protein or the adaptor protein SLP-76. Our data elucidate the mechanism of SLP-65 membrane recruitment and suggest that leucine zipper motifs are essential interaction domains of signaling proteins

Imaging Spatiotemporal Activities of ZAP-70 in Live T Cells Using a FRET-Based Biosensor

International audienceThe zeta-chain-associated protein kinase 70 kDa (ZAP-70), a member of the spleen tyrosine kinase (Syk) family, plays an essential role in early T cell receptor (TCR) signaling. Defects in ZAP-70 lead to impaired thymocyte development and peripheral T cell activation. To better understand its activation dynamics and regulation, we visualized ZAP-70 activities in single live T cells with a Forster resonance energy transfer (FRET)aEurobased biosensor, which was designed for probing kinase activities of the Syk family. We observed in Jurkat E6.1 T cells rapid and specific FRET changes following anti-CD3 stimulation and subsequent piceatannol inhibition. The initiation of ZAP-70 activation was prompt (within 10 s) and correlates with the accompanied intracellular calcium elevation, as revealed by simultaneous imaging of the biosensor and calcium. Different from the previously reported ZAP-70 activation in the immunological synapse and the opposite pole (anti-synapse), we have observed rapid and sustained ZAP-70 activation only at the synapse with superantigen-pulsed Raji B cells. Furthermore, ZAP-70 signaling was impaired by cholesterol depletion, further supporting the importance of membrane organization in TCR signaling. Together our results provide a direct characterization of the spatiotemporal features of ZAP-70 activity in real time at subcellular levels

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

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