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...

Ras-GTP imaging studies using the Ras-binding domain (RBD) of the Ras effector c-Raf as a reporter for overexpressed Ras have produced discrepant results about the possible activation of Ras at the Golgi apparatus. We report that RBD oligomerization provides probes for visualization of endogenous Ras-GTP, obviating Ras overexpression and the side effects derived thereof. RBD oligomerization results in tenacious binding to Ras-GTP and interruption of Ras signalling. Trimeric RBD probes fused to green fluorescent protein report agonist-induced endogenous Ras activation at the plasma membrane (PM) of COS-7, PC12 and Jurkat cells, but do not accumulate at the Golgi. PM illumination is exacerbated by Ras overexpression and its sensitivity to dominant-negative RasS17N and pharmacological manipulations matches Ras-GTP formation assessed biochemically. Our data illustrate that endogenous Golgi-located Ras is not under the control of growth factors and argue for the PM as the predominant site of agonist-induced Ras activation.

No abstract

Signaling of receptor tyrosine kinases (RTKs) is regulated by protein-tyrosine phosphatases (PTPs). We previously discovered the efficient downregulation of Ros RTK signaling by the SH2 domain PTP SHP-1, which involves a direct interaction of both molecules. Here, we studied the mechanism of this interaction in detail. Phosphopeptides representing the SHP-1 candidate binding sites in the Ros cytoplasmic domain, pY2267 and pY2327, display high affinity binding to the SHP-1 N-terminal SH2 domain (Kd=217 nM and 171 nM, respectively). Y2327 is, however, a poor substrate of Ros kinase and, therefore, contributes little to SHP-1 binding in vitro. To explore the mechanism of association in intact cells, functional fluorescent fusion proteins of Ros and SHP-1 were generated. Complexes of both molecules could be detected by Förster resonance energy transfer (FRET) in intact HEK293 and COS7 cells. As expected, the association required the functional SHP-1 N-terminal SH2 domain. Unexpectedly, pY2267 and pY2327 both contributed to the association. Mutation of Y2327 reduced constitutive association in COS7 cells. Ligand-dependent association was abrogated upon mutation of Y2267 but remained intact when Y2327 was mutated. A phosphopeptide representing the binding site pY2267 was a poor substrate for SHP-1, whereas Ros activation loop phosphotyrosines were effectively dephosphorylated. We propose a model for SHP-1-Ros interaction in which ligand-stimulated phosphorylation of Ros Y2267 by Ros, phosphorylation of Y2327 by a heterologous kinase, and inactivation of Ros by SHP-1-mediated dephosphorylation play a role in the regulation of complex stability.

Trafficking of malignant lymphocytes is fundamental to the biology of chronic lymphocytic leukemia (CLL). Transendothelial migration (TEM) of normal lymphocytes into lymph nodes requires the chemokine-induced activation of Rap1 and αLβ2 integrin. However, in most cases of CLL, Rap1 is refractory to chemokine stimulation, resulting in failed αLβ2 activation and TEM unless α4β1 is coexpressed. In this study, we show that the inability of CXCL12 to induce Rap1 GTP loading in CLL cells results from failure of Rap1-containing endosomes to translocate to the plasma membrane. Furthermore, failure of chemokine-induced Rap1 translocation/GTP loading was associated with a specific pattern of cellular IgD distribution resembling that observed in normal B cells anergized by DNA-based Ags. Anergic features and chemokine unresponsiveness could be simultaneously reversed by culturing CLL cells ex vivo, suggesting that these two features are coupled and driven by stimuli present in the in vivo microenvironment. Finally, we show that failure of Rap1 translocation/GTP loading is linked to defective activation of phospholipase D1 and its upstream activator Arf1. Taken together, our findings indicate that chemokine unresponsiveness in CLL lymphocytes results from failure of Arf1/phospholipase D1–mediated translocation of Rap1 to the plasma membrane for GTP loading and may be a specific feature of anergy induced by DNA Ags.