Mira Barda-Saad

Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1.

Receptor oligomerization is vital for activating intracellular signaling, in part by initiating events that recruit effector and adaptor proteins to sites of active signaling. Whether these distal molecules themselves oligomerize is not well appreciated. In this study, we examined the molecular interactions of the adaptor protein GRB2. In T cells, the SH2 domain of GRB2 binds phosphorylated tyrosines on the adaptor protein LAT and the GRB2 SH3 domains associate with the proline-rich regions of SOS1 and CBL. Using biochemical and biophysical techniques in conjunction with confocal microscopy, we observed that the simultaneous association of GRB2, via its SH2 and SH3 domains, with multivalent ligands led to the oligomerization of these ligands, which affected signaling. These data suggest that multipoint binding of distal adaptor proteins mediates the formation of oligomeric signaling clusters vital for intracellular signaling.

Recruitment and activation of PLCγ1 in T cells: a new insight into old domains

Engagement of the T‐cell antigen receptor leads to recruitment of phospholipase Cγ1 (PLCγ1) to the LAT‐nucleated signaling complex and to PLCγ1 activation in a tyrosine phosphorylation‐dependent manner. The mechanism of PLCγ1 recruitment and the role of PLCγ1 Src homology (SH) domains in this process remain incompletely understood. Using a combination of biochemical methods and real‐time fluorescent imaging, we show here that the N‐terminal SH2 domain of PLCγ1 is necessary but not sufficient for its recruitment. Either the SH3 or C‐terminal SH2 domain of PLCγ1, with the participation of Vav1, c‐Cbl and Slp76, are required to stabilize PLCγ1 recruitment. All three PLCγ1 SH domains are required for phosphorylation of PLCγ1 Y783, which is critical for enzyme activation. These novel findings entailed revision of the currently accepted model of PLCγ1 recruitment and activation in T lymphocytes.

Early phosphorylation kinetics of proteins involved in proximal TCR-mediated signaling pathways.

Activation of T cells via the stimulation of the TCR plays a central role in the adaptive immunological response. Although much is known about TCR-stimulated signaling pathways, there are still gaps in our knowledge about the kinetics and sequence of events during early activation and about the in vivo specificity of kinases involved in these proximal signaling pathways. This information is important not only for understanding the activation of signaling pathways important for T cell function but also for the development of drug targets and computer-based molecular models. In this study, phospho-specific Abs directed toward individual sites on signaling proteins were used to investigate the early phosphorylation kinetics of proteins involved in proximal TCR-induced pathways. These studies indicate that linker for activation of T cells’ tyrosines have substantially different phosphorylation kinetics and that Src homology 2 domain-containing leukocyte protein of 76 kDa has rapid, transient phosphorylation kinetics compared to other proteins. In additions, we provide evidence that ZAP-70 is the primary in vivo kinase for LAT tyrosine 191 and that Itk plays a role in the phosphorylation of tyrosine 783 on phospholipase C-γ1. In total, these studies give new insight into the sequence, kinetics and specificity of early TCR-mediated signaling events that are vital for T cell activation.

Cooperative interactions at the SLP-76 complex are critical for actin polymerization

T‐cell antigen receptor (TCR) engagement induces formation of multi‐protein signalling complexes essential for regulating T‐cell functions. Generation of a complex of SLP‐76, Nck and VAV1 is crucial for regulation of the actin machinery. We define the composition, stoichiometry and specificity of interactions in the SLP‐76, Nck and VAV1 complex. Our data reveal that this complex can contain one SLP‐76 molecule, two Nck and two VAV1 molecules. A direct interaction between Nck and VAV1 is mediated by binding between the C‐terminal SH3 domain of Nck and the VAV1 N‐terminal SH3 domain. Disruption of the VAV1:Nck interaction deleteriously affected actin polymerization. These novel findings shed new light on the mechanism of actin polymerization after T‐cell activation.

c-Cbl-Mediated Regulation of LAT-Nucleated Signaling Complexes

ABSTRACT The engagement of the T-cell receptor (TCR) causes the rapid recruitment of multiple signaling molecules into clusters with the TCR. Upon receptor activation, the adapters LAT and SLP-76, visualized as chimeric proteins tagged with yellow fluorescent protein, transiently associate with and then rapidly dissociate from the TCR. Previously, we demonstrated that after recruitment into signaling clusters, SLP-76 is endocytosed in vesicles via a lipid raft-dependent pathway that requires the interaction of the endocytic machinery with ubiquitylated proteins. In this study, we focus on LAT and demonstrate that signaling clusters containing this adapter are internalized into distinct intracellular compartments and dissipate rapidly upon TCR activation. The internalization of LAT was inhibited in cells expressing versions of the ubiquitin ligase c-Cbl mutated in the RING domain and in T cells from mice lacking c-Cbl. Moreover, c-Cbl RING mutant forms suppressed LAT ubiquitylation and caused an increase in cellular LAT levels, as well as basal and TCR-induced levels of phosphorylated LAT. Collectively, these data indicate that following the rapid formation of signaling complexes upon TCR stimulation, c-Cbl activity is involved in the internalization and possible downregulation of a subset of activated signaling molecules.

T‐Cell Antigen Receptor‐Induced Signaling Complexes: Internalization Via a Cholesterol‐Dependent Endocytic Pathway

T‐cell antigen receptor engagement causes the rapid assembly of signaling complexes. The adapter protein SLP‐76, detected as SLP‐yellow fluorescent protein, initially clustered with the TCR and other proteins, then translocated medially on microtubules. As shown by total internal reflection fluorescence microscopy and the inhibition of SLP‐76 movement at 16°C, this movement required endocytosis. Immunoelectron microscopy showed SLP‐76 staining of smooth pits and tubules. Cholesterol depletion decreased the movement of SLP‐76 clusters, as did coexpression of the ubiquitin‐interacting motif domain from eps15. These data are consistent with the internalization of SLP‐76 via a lipid raft‐dependent pathway that requires interaction of the endocytic machinery with ubiquitinylated proteins. The endocytosed SLP‐76 clusters contained phosphorylated SLP‐76 and phosphorylated LAT. The raft‐associated, transmembrane protein LAT likely targets SLP‐76 to endocytic vesicles. The endocytosis of active SLP‐76 and LAT complexes suggests a possible mechanism for downregulation of signaling complexes induced by TCR activation.

Role of activin A in negative regulation of normal and tumor B lymphocytes

Activin A, a member of the transforming growth factor β superfamily, has a wide spread expression pattern and pleiotropic functions. In thisoverview we summarize data that points to a role of activin A innegative regulation of B lineage lymphocytes. Experiments performed byus and by other groups revealed the capacity of activin A to causeapoptotic death of tumor myeloma cells, through mechanisms of cellcycle inhibition and antagonism with the survival signal ofinterleukin‐6. In vitro studies on B lymphocyte generation from bonemarrow stem cells and use of human nasal polyps as a model of inflamedtissue further demonstrate an inhibitory role of activin A in B cellspread and accumulation. These data are analyzed with respect to ourmodel of tissue organization that we term the “restrictin model ofcell growth regulation.” This model assumes a morphogen‐like role ofactivin A in the hematopoietic system. Thus, the relative concentrationof biologically functional activin A, in different parts of the tissue, may determine the local B cell content and functional state of thesecells within a specific microenvironment.

Functional cooperation between the proteins Nck and ADAP is fundamental for actin reorganization.

ABSTRACT T cell antigen receptor (TCR) activation triggers profound changes in the actin cytoskeleton. In addition to controlling cellular shape and polarity, this process regulates vital T cell responses, such as T cell adhesion, motility, and proliferation. These depend on the recruitment of the signaling proteins Nck and Wiskott-Aldrich syndrome protein (WASp) to the site of TCR activation and on the functional properties of the adapter proteins linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76). We now demonstrate that Nck is necessary but insufficient for the recruitment of WASp. We show that two pathways lead to SLP76-dependent actin rearrangement. One requires the SLP76 acidic domain, crucial to association with the Nck SH2 domain, and another requires the SLP76 SH2 domain, essential for interaction with the adhesion- and degranulation-promoting adapter protein ADAP. Functional cooperation between Nck and ADAP mediates SLP76-WASp interactions and actin rearrangement. We also reveal the molecular mechanism linking ADAP to actin reorganization.

The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Examining multiprotein signaling complexes from all angles: The use of complementary techniques to characterize complex formation at the adapter protein, linker for activation of T cells

Dynamic protein–protein interactions are involved in most physiological processes and, in particular, for the formation of multiprotein signaling complexes at transmembrane receptors, adapter proteins and effector molecules. Because the unregulated induction of signaling complexes has substantial clinical relevance, the investigation of these complexes is an active area of research. These studies strive to answer questions about the composition and function of multiprotein signaling complexes, along with the molecular mechanisms of their formation. In this review, the adapter protein, linker for activation of T cells (LAT), will be employed as a model to exemplify how signaling complexes are characterized using a range of techniques. The intensive investigation of LAT highlights how the systematic use of complementary techniques leads to an integrated understanding of the formation, composition and function of multiprotein signaling complexes that occur at receptors, adapter proteins and effector molecules.