The ADAP/SKAP55 Signaling Module Regulates T-Cell Receptor-Mediated Integrin Activation through Plasma Membrane Targeting of Rap1

Abstract

Adhesion of T cells after stimulation of the T-cell receptor (TCR) is mediated via signaling processes that have collectively been termed inside-out signaling. The molecular basis for inside-out signalingis not yet completely understood. Here, we show that a signaling module comprising the cytosolic adapter proteins ADAP and SKAP55 is involved in TCR-mediated inside-out signaling and, moreover, that the interaction between ADAP and SKAP55 is mandatory for integrin activation. Disruption of the ADAP/SKAP55 module leads to displacement of the small GTPase Rap1 from the plasma membrane without influencing its GTPase activity. These findings suggest that the ADAP/SKAP55 complex serves to recruit activated Rap1 to the plasma membrane. In line with this hypothesis is the finding that membrane targeting of the ADAP/SKAP55 module induces T-cell adhesion in the absence of TCR-mediated stimuli. However, it appears as if the ADAP/SKAP55 module can exert its signaling function outside of the classical raft fraction of the cell membrane.Within the immune system, integrins play important roles in regulating the interaction of T cells with other cells and with proteins of the extracellular matrix. By mediating T-cell adhesion, integrins control the homing and the trafficking of T cells as well as the interaction between T cells and antigen-presenting cells (34, 41). The major integrins expressed on T cells are the ␤2-integrin LFA-1 (␣L␤2) as well as members of the ␤1-family of integrins (␣4␤1, ␣5␤1, ␣6␤1, and VLA) (25). The physiologic ligands of LFA-1 include the intercellular adhesion molecule 1 (ICAM-1), ICAM-2, and ICAM-3 (25), whereas ligands for ␤1-integrins are vascular cell adhesion molecule 1 (VCAM-1) or proteins of the extracellular matrix, such as fibronectin (13,54).On resting T cells, ␤1-and ␤2-integrins are expressed in an inactive state. However, ligation of the T-cell receptor (TCR) by antigen/major histocompatibility complexes results in a rapid increase in the activity of ␤1-and ␤2-integrins, thereby enhancing ligand binding (15,46,50). Two distinct mechanisms mediate the activation of integrins. First, the affinity of an integrin for its ligand is enhanced, and second, the lateral mobility becomes altered, which results in integrin clustering (avidity regulation) (14). The processes leading to the activation of integrins have collectively been termed inside-out signaling (14, 15, 28).Several molecules have been suggested to play critical roles during TCR-mediated activation of ␤1-and ␤2-integrins (14, 28). Among these is the small GTPase Rap1, whose role for integrin activation has been a matter of intense research during the last few years (4, 29). The mechanisms for how Rap1 becomes activated are not yet completely understood (4). Rap1 activation has been shown to be mediated by particular guanine nucleotide exchange factors (GEFs), such as C3G, and Epac (5,8,11). It has been proposed that Rap1 is associated with CalDAG-GEFI and that TCR-induced Rap1 activation is dependent upon the activation of phosphol...

HPK1 competes with ADAP for SLP‐76 binding and via Rap1 negatively affects T‐cell adhesion

“…Two additional adapter molecules, adhesion and degranulation promoting adaptor protein (ADAP) and 55-kDa src kinase associated phosphoprotein (SKAP-55), are recruited upon TCR-mediated phosphorylation of ADAP on Y 595 DDV or Y 651 DDV to the SH2-domain of SLP-76 [3,4]. ADAP and SKAP-55 form a trimolecular complex with RIAM (Ras-related protein 1 (Rap1)-GTP interact-ing adapter molecule) which binds active Rap1 and thereby facilitates TCR-mediated integrin activation [5,6]. In addition, the Rap1-GTP effector regulator for cell adhesion and polarization enriched in lymphoid tissues (RapL) was recently shown to directly bind SKAP-55, which might differentially influence integrin affinity/avidity regulation [7].…”

“…ing adapter molecule) which binds active Rap1 and thereby facilitates TCR-mediated integrin activation [5,6]. In addition, the Rap1-GTP effector regulator for cell adhesion and polarization enriched in lymphoid tissues (RapL) was recently shown to directly bind SKAP-55, which might differentially influence integrin affinity/avidity regulation [7].…”

In vivo disruption of T cell development by expression of a dominant‐negative polypeptide designed to abolish the SLP‐76/Gads interaction

“…ADAP itself has been directly linked to integrin activation, as ADAP -/-T cells do not adhere to fibronectin, ICAM, or VCAM following TCR ligation [31,32]. One model for how a SLP-76/ ADAP association may lead to integrin activation is through Src kinase-associated phosphoprotein of 55 kDa, an adaptor that is constitutively associated with ADAP [52] and has been linked to TCR-induced integrin activation [53,54].Other signaling proteins involved in TCR-induced integrin activation have been identified, but details of the pathway are not yet clearly defined. One such protein is the small GTPase Rap1.…”

“…Cells were cultured at 37 C for 3-6 h, at which time the medium was replaced with fresh culture medium. The generation of soluble ICAM-1 was previously described [54]. …”

ARAP, a Novel Adaptor Protein, Is Required for TCR Signaling and Integrin-Mediated Adhesion

“…SKAP1 plays a central role in ADAP-mediated signaling to integrins, and the identical motifs of the ARAP PR region occur in the primary site of mouse and human ADAP that binds to SKAP1 (36,37). Therefore, we examined whether ARAP also binds to SKAP1 through the ARAP PR region.…”