The ␤2-integrin lymphocyte functionassociated antigen-1 (LFA-1) plays a crucial role within the immune system. It regulates the interaction between T cells and antigen-presenting cells and facilitates T-cell adhesion to the endothelium, a process that is important for lymphocyte extravasation and homing. Signals mediated via the T-cell receptor and the chemokine receptor CCR7 activate LFA-1 through processes known as inside-out signaling. The molecular mechanisms underlying inside-out signaling are not completely understood. Here, we have assessed the role of the ADAP/SKAP55 module for CCR7-mediated signaling. We show that loss of the module delays homing and reduces intranodal T-cell motility in vivo. This is probably because of a defect in CCR7-mediated adhesion that affects both affinity and avidity regulation of LFA-1. Further analysis of how the ADAP/SKAP55 module regulates CCR7-induced integrin activation revealed that 2 independent pools of the module are expressed in T cells. One pool interacts with a RAPL/Mst1 complex, whereas the other pool is linked to a RIAM/Mst1/Kindlin-3 complex. Importantly, both the RAPL/Mst1 and the RIAM/Mst1/Kindlin-3 complexes require ADAP/SKAP55 for binding to LFA-1 upon CCR7 stimulation. Hence, 2 independent ADAP/SKAP55 modules are essential components of the signaling machinery that regulates affinity and avidity of LFA-1 in response to CCR7. IntroductionWithin the immune system, the ␤2-integrin LFA-1 (lymphocyte function-associated antigen; ␣L␤2) mediates T-cell adhesion to the endothelium, homing of T cells to secondary lymphoid organs, and T-cell activation through the interaction of lymphocytes with antigen-presenting cells (APCs). 1,2 On resting T cells, LFA-1 is expressed in an inactive form and adopts a low-affinity conformation for its ligands, the ICAM molecules (inter cell adhesion molecules). 3,4 Triggering of the T-cell receptor (TCR) by Ag/MHC complexes or of the chemokine receptor CCR7 by CCL21 induces a conformational change of LFA-1 that increases its affinity for ICAM-1 (affinity regulation). Furthermore, these stimuli also facilitate clustering of LFA-1, a process termed avidity modulation. 3,4 The molecular events leading to LFA-1 activation have collectively been termed "inside-out signaling."Research of the last decade has revealed that the small GTPase Rap1, the 2 Rap1 effector proteins RIAM (Rap1 interacting adapter molecule) and RAPL (regulator for cell adhesion and polarization enriched in lymphoid tissues), and the RAPL-interacting mammalian Ste20-like kinase (Mst1) are critically involved in TCR-and CCR7-mediated signaling events regulating T-cell adhesion, LFA-1 affinity and avidity modulation, and T-cell-APC interactions. 3,4 Indeed, RAPL-and Mst1-deficient T cells show defects in adhesion, homing, and intranodal migration in vivo. [5][6][7] In addition to RAPL, RIAM, and Mst1, the cytosolic adapter proteins ADAP (adhesion and degranulating promoting adapter protein), and SKAP55 (Src-kinase associated phosphoprotein of 55 kDa) are crucial for i...

T cells are key mediators of cell-mediated immunity. Their functions and proliferation result from T cell-specific receptor signaling (TCR/CD28) that activates the NF-κB, NFAT, Ras-MAPK, and PI3K-Akt pathways. Their development and activation also involve a complex array of signaling pathways that regulate gene expression networks, including signaling of mTOR, Notch, Wnt, Hedgehog, TGF-β, and toll-like receptors. Furthermore, recent discoveries have provided two molecular hallmarks of potential generality: miRNA patterns and polycomb-mediated epigenetic reprogramming, which can strongly coordinate the balance between molecular networks in lymphocytes. Their deregulation apparently causes T cell disorders, such as T cell acute lymphoblastic leukemia (T-ALL), and human T cell leukemia virus (HTLV-1)-induced adult T cell leukemia (ATL). This review continues with a description of our understanding of crosstalk among the signaling pathways, which contribute to the highly orchestrated development of T cell fate specification under both normal physiological and pathological conditions.

The adaptor protein Src homology 2 (SH2) domain containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adaptor proteins that function together to activate second messengers required for TCR signal propagation. The N-terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with the adhesion- and degranulation-promoting adaptor protein (ADAP) and hematopoietic progenitor kinase 1 (HPK1). Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production and phosphorylation of protein kinase D (PKD) and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells.

The monoclonal antibody B13-DE1 binds fluorescein, several fluorescein derivatives, and three peptide mimotopes. Our results revealed that this antibody also catalyzed the redox reaction of resazurin to resorufin, which are both structurally related to fluorescein. By using sodium sulfite as a reducing agent, the antibody B13-DE1 lowered the activation energy of this reaction. The Michaelis-Menten constant and turnover number of the catalyzed reaction were determined as 4.2 µmol/l and 0.0056 s(-1) , respectively. Because the results showed that fluorescein inhibited the catalytic activity of the antibody, we assume that the antigen-binding site and the catalytic active site are identical.

Intrinsically disordered proteins (IDPs) play a vital role in biological processes that rely on transient molecular compartmentation1. In T cells, the dynamic switching between migration and adhesion mandates a high degree of plasticity in the interplay of adhesion and signaling molecules with the actin cytoskeleton2,3. Here, we show that the N-terminal intrinsically disordered region (IDR) of adhesion- and degranulation-promoting adapter protein (ADAP) acts as a multipronged scaffold for G- and F-actin, thereby promoting actin polymerization and bundling. Positively charged motifs, along a sequence of at least 200 amino acids, interact with both longitudinal sides of G-actin in a promiscuous manner. These polymorphic interactions with ADAP become constrained to one side once F-actin is formed. Actin polymerization by ADAP acts in synergy with a capping protein but competes with cofilin. In T cells, ablation of ADAP impairs adhesion and migration with a time-dependent reduction of the F-actin content in response to chemokine or T cell receptor (TCR) engagement. Our data suggest that IDR-assisted molecular crowding of actin above the critical concentration defines a new mechanism to regulate cytoskeletal dynamics. The principle of IDRs serving as molecular sponges to facilitate regulated self-assembly of filament-forming proteins might be a general phenomenon.