And Degranulation-promoting Adapter Protein Research Articles

Regulation and Function of SKAP-55 Non-canonical Motif Binding to the SH3c Domain of Adhesion and Degranulation-promoting Adaptor Protein

The immune cell adaptor adhesion and degranulation promoting adaptor protein (ADAP) and its binding to T-cell adaptor Src kinase-associated protein of 55 kDa (SKAP-55) play a key role in the modulation of T-cell adhesion. While primary binding occurs via SKAP-55 SH3 domain binding to a proline-rich region in ADAP, a second interaction occurs between the ADAP C-terminal SH3 domain (ADAP-SH3c) and a non-canonical RKXXY294XXY297 motif in SKAP-55. Increasing numbers of non-canonical SH3 domain binding motifs have been identified in a number of biological systems. The presence of tyrosine residues in the SKAP-55 RKXXY294XXY297 motif suggested that phosphorylation might influence this unusual SH3 domain interaction. Here, we show that the Src kinase p59fyn can induce the in vivo phosphorylation of the motif, and this event blocks ADAP-SH3c domain binding to the peptide motif. The importance of tyrosine phosphorylation was confirmed by plasmon resonance interaction analysis showing that phosphorylation of Tyr294 residue plays a central role in mediating dissociation, whereas phosphorylation of the second Tyr297 had no effect. Although loss of this secondary interaction did not result in the disruption of the complex, the Y294F mutation blocked T-cell receptor-induced up-regulation of lymphocyte function-associated antigen-1-mediated adhesion to intercellular adhesion molecule-1 and interleukin-2 promoter activity. Our findings identify a RKXXY294 motif in SKAP-55 that mediates unique ADAP SH3c domain binding and is needed for LFA-1-mediated adhesion and cytokine production.

Association of c-SRC and adhesion and degranulation-promoting adaptor protein (ADAP) in osteoclastogenesis in vitro

Association of c-SRC and adhesion and degranulation-promoting adaptor protein (ADAP) in osteoclastogenesis in vitro

Physical and Functional Association of c-Src and Adhesion and Degranulation Promoting Adaptor Protein (ADAP) in Osteoclastogenesis in Vitro

c-Src plays a crucial role in osteoclastogenesis. In this study, we searched for c-Src-binding proteins using a combination of pull-down assays and mass spectrometric analysis, and identified the association of adhesion and degranulation promoting adaptor protein (ADAP) with c-Src in RAW264 cells and osteoclast precursors prepared from bone marrow cells. The kinase activity and the SH2 domain of c-Src were required for this association and Tyr807 in the extreme carboxyl terminus of ADAP was identified as a major recognition site. ADAP was found to be expressed in cells at the prefusion stage and localized mainly in the leading edge of lamellipodia and in pseudopodia. Tyrosine phosphorylation of ADAP was induced in an integrin-dependent manner, and the level was Src kinase-dependent. ADAP-knockdown RAW264 cells showed retarded migration and formed few multinucleated cells. Cas, known to be phosphorylated by c-Src, was identified as a major tyrosine-phosphorylated protein in differentiating RAW264 cells and the phosphorylation appeared to be decreased in ADAP-knockdown cells. ADAP thus may play an important role as a partner of c-Src for cell migration and progression to the multinucleated cell stage in osteoclastogenesis in vitro.

The Helically Extended SH3 Domain of the T Cell Adaptor Protein ADAP is a Novel Lipid Interaction Domain

Adhesion and degranulation-promoting adapter protein (ADAP) is critically involved in downstream signalling events triggered by the activation of the T cell receptor. Cytokine production, proliferation and integrin clustering of T cells are dependent on ADAP function, but the molecular basis for these processes is poorly understood. We now show the hSH3 domain of ADAP to be a lipid-interaction module that binds to acidic lipids, including phosphatidylinositides. Positively charged surface patches of the domain preferentially bind to polyvalent acidic lipids such as PIP2 or PIP3 over the monovalent PS phospholipid and this interaction is dependent on the N-terminal helix of the hSH3 domain fold. Basic amino acid side-chains from the SH3 scaffold also contribute to lipid binding. In the context of T cell signalling, our findings suggest that ADAP, upon recruitment to the cell-cell junction as part of a multiprotein complex, directly interacts with phosphoinositide-enriched regions of the plasma membrane. Furthermore, the ADAP lipid interaction defines the helically extended SH3 scaffold as a novel member of membrane interaction domains.

ADAP-SLP-76 binding differentially regulates supramolecular activation cluster (SMAC) formation relative to T cell-APC conjugation.

T cell–APC conjugation as mediated by leukocyte function-associated antigen-1 (LFA-1)–intercellular adhesion molecule (ICAM)-1 binding is followed by formation of the supramolecular activation cluster (SMAC) at the immunological synapse. The intracellular processes that regulate SMAC formation and its influence on T cell function are important questions to be addressed. Here, using a mutational approach, we demonstrate that binding of adaptor adhesion and degranulation promoting adaptor protein (ADAP) to SLP-76 differentially regulates peripheral SMAC (pSMAC) formation relative to conjugation. Although mutation of the YDDV sites (termed M12) disrupted SLP-76 SH2 domain binding and prevented the ability of ADAP to increase conjugation and LFA-1 clustering, M12 acted selectively as a dominant negative (DN) inhibitor of pSMAC formation, an effect that was paralleled by a DN effect on interleukin-2 production. ADAP also colocalized with LFA-1 at the immunological synapse. Our findings identify ADAP–SLP-76 binding as a signaling event that differentially regulates SMAC formation, and support a role for SMAC formation in T cell cytokine production.

Differential requirement for adapter proteins Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and adhesion- and degranulation-promoting adapter protein in FcepsilonRI signaling and mast cell function.

The adapter molecule Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is essential for FcepsilonRI-mediated signaling, degranulation and IL-6 production in mast cells. To test the structural requirements of SLP-76 in mast cell signaling and function, we have studied the functional responses of murine bone marrow-derived mast cells (BMMCs) expressing mutant forms of SLP-76. We found that the N-terminal tyrosines as well as the central proline-rich region of SLP-76 are required for participation of SLP-76 in FcepsilonRI-mediated signaling and function. The C-terminal SH2 domain of SLP-76 also contributes to optimal function of SLP-76 in mast cells. Another adapter molecule, adhesion- and degranulation-promoting adapter protein (ADAP), is known to bind the SH2 domain of SLP-76, and cell line studies have implicated ADAP in mast cell adhesion and FcepsilonRI-induced degranulation. Surprisingly, we found that mast cells lacking ADAP expression demonstrate no defects in FcepsilonRI-induced adhesion, granule release, or IL-6 production, and that ADAP-deficient mice produce a normal passive systemic anaphylactic response. Thus, failure to bind ADAP does not underlie the functional defects exhibited by SLP-76 SH2 domain mutant-expressing mast cells.

Positive and negative adaptors in T-cell signalling.

Positive and negative adaptors in T-cell signalling.

The TCR ADAPts to integrin-mediated cell adhesion.

Adapter proteins regulate leukocyte signal transduction through recruitment of effector molecules to multiprotein complexes. Recent studies in Adhesion and Degranulation promoting Adapter Protein (ADAP)-deficient mice have established that the cytoplasmic phosphoprotein ADAP is required for optimal, mature T-cell proliferation. Furthermore, ADAP plays a key role in T-cell antigen receptor (TCR)-mediated 'inside out' signaling leading to integrin activation and to enhanced cellular adhesion to integrin ligands. ADAP associates physically with molecules known to play roles in the regulation of TCR-stimulated actin polymerization. These associations support the hypothesis that ADAP functions in actin cytoskeletal reorganization leading to cellular adhesion and activation.

SKAP-55 regulates integrin adhesion and formation of T cell-APC conjugates.

Src kinase-associated phosphoprotein of 55 kDa (SKAP-55; encoded by SCAP1) is a T cell adaptor protein of unknown function that contains a pleckstrin homology and an SH3 domain. Here we show that SKAP-55 regulates integrin-mediated adhesion and conjugate formation between T cells and antigen-presenting cells (APCs). SKAP-55 enhances adhesion to fibronectin and intercellular adhesion molecule-1 (ICAM-1), colocalizes with actin at the T cell-APC synapse and promotes the clustering of lymphocyte-associated antigen-1 (LFA-1). Enhanced conjugation is comparable to that induced by adhesion and degranulation-promoting adaptor protein (ADAP), a binding partner of SKAP-55, and is abrogated by deletion of the SKAP-55 SH3 domain. Conjugate formation is accompanied by the translocation of SKAP-55 to membrane rafts, an event that is regulated by both LFA-1 and T cell receptor ligation. Our findings identify a mechanism by which SKAP-55 modulates T cell responses to antigen.

ADAP-ting TCR signaling to integrins.

Adaptor proteins are essential components of T cell receptor (TCR) signaling cascades regulating gene transcription and cytoskeletal reorganization. The molecular adaptor adhesion- and degranulation-promoting adaptor protein (ADAP), also known as Fyn binding protein (FYB) or Slp-76-associated protein of 130 kilodaltons (SLAP-130), interacts with a number of signaling intermediates including Slp-76, the Src family tyrosine kinase Fyn, vasodilator-stimulated phosphoprotein (VASP), and the actin-nucleating protein WASP. Recently ADAP was shown genetically to positively regulate T cell activation, TCR-induced integrin clustering, and T cell adhesion. The mechanism by which ADAP couples TCR stimulation to integrin clustering remains unclear; however, studies of ADAP, the exchange factor Vav1, and WASP suggest that TCR and integrin clustering may be controlled by distinct signaling pathways.