Barak Reicher

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.

Ubiquitylation-Dependent Negative Regulation of WASp Is Essential for Actin Cytoskeleton Dynamics

ABSTRACT The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin dynamics during cell motility and adhesion, and mutations in its gene are responsible for Wiskott-Aldrich syndrome (WAS). Here, we demonstrate that WASp is ubiquitylated following T-cell antigen receptor (TCR) activation. WASp phosphorylation at tyrosine 291 results in recruitment of the E3 ligase Cbl-b, which, together with c-Cbl, carries out WASp ubiquitylation. Lysine residues 76 and 81, located at the WASp WH1 domain, which contains the vast majority of WASp gene mutations, serve as the ubiquitylation sites. Disruption of WASp ubiquitylation causes WASp accumulation and alters actin dynamics and the formation of actin-dependent structures. Our data suggest that regulated degradation of activated WASp might be an efficient strategy by which the duration and localization of actin rearrangement and the intensity of T-cell activation are controlled.

Wiskott–Aldrich syndrome protein – dynamic regulation of actin homeostasis: from activation through function and signal termination in T lymphocytes

The actin cytoskeleton network forms a key link between T‐cell antigen receptor (TCR) stimulation and T‐cell effector functions, providing a structural basis for T‐cell morphological changes and signal transduction. Accumulating evidence positions the Wiskott–Aldrich syndrome protein (WASp), a scaffolding protein that promotes actin polymerization, at the center of actin cytoskeleton‐dependent T‐cell function. During the past decade, we and others have utilized multidisciplinary technologies, including live‐cell imaging, biochemical, and biophysical analyses, to gain insight into the mechanisms by which WASp and other cytoskeletal proteins control actin homeostasis. Following TCR engagement, WASp is rapidly activated and recruited to TCR microclusters, as part of multiprotein complexes, where it promotes actin remodeling. Late in the activation process, WASp is internalized and eventually degraded. In this review, we describe the dynamic interactions of WASp with signaling proteins, which regulate its activation and recruitment to the TCR and to actin‐rich sites. Finally, we present the molecular mechanism of WASp downregulation. Some of the signaling proteins that mediate WASp activation eventually lead to its degradation. Thus, we focus here on the regulation of WASp expression and function and the mechanisms whereby they control actin machinery and T‐cell effector functions.

Studying the Dynamics of SLP-76, Nck, and Vav1 Multimolecular Complex Formation in Live Human Cells with Triple-Color FRET

The formation of a multiprotein complex in response to T cell activation is monitored in live cells. Watching Protein Complex Assembly In response to the activation of the T cell receptor (TCR) on T cells, various kinases, adaptors, and signaling proteins assemble to form various multimolecular complexes to mediate T cell activation. The complex containing the adaptor proteins SLP-76 and Nck and the guanine nucleotide exchange factor Vav1 is required for the cytoskeletal rearrangements that occur in response to TCR stimulation. Pauker et al. developed an imaging technique (involving triple-color FRET analysis) to monitor the dynamics of the various interactions between SLP-76, Nck, and Vav1 in stimulated live T cells and observed formation of the tripartite complex. They found that Nck and Vav1 were preassembled as heterodimers before TCR stimulation and that mutation of a critical residue in Vav1 disrupted actin rearrangement. This study establishes the use of this technique in the investigation of other multiprotein complexes in live cells. Protein-protein interactions regulate and control many cellular functions. A multimolecular complex consisting of the adaptor proteins SLP-76 (Src homology 2 domain–containing leukocyte protein of 76 kD), Nck, and the guanine nucleotide exchange factor Vav1 is recruited to the T cell side of the interface with an antigen-presenting cell during initial T cell activation. This complex is crucial for regulation of the actin machinery, antigen recognition, and signaling in T cells. We studied the interactions between these proteins as well as the dynamics of their recruitment into a complex that governs cytoskeletal reorganization. We developed a triple-color Förster resonance energy transfer (3FRET) system to observe the dynamics of the formation of this trimolecular signaling complex in live human T cells and to follow the three molecular interactions in parallel. Using the 3FRET system, we demonstrated that dimers of Nck and Vav1 were constitutively formed independently of both T cell activation and the association between SLP-76 and Nck. After T cell receptor stimulation, SLP-76 was phosphorylated, which enabled the binding of Nck. A point mutation in the proline-rich site of Vav1, which abolishes its binding to Nck, impaired actin rearrangement, suggesting that Nck-Vav1 dimers play a critical role in regulation of the actin machinery. We suggest that these findings revise the accepted model of the formation of a complex of SLP-76, Nck, and Vav1 and demonstrate the use of 3FRET as a tool to study signal transduction in live cells.

Multiple pathways leading from the T-cell antigen receptor to the actin cytoskeleton network

Dynamic rearrangements of the actin cytoskeleton, following T‐cell antigen receptor (TCR) engagement, provide the structural matrix and flexibility to enable intracellular signal transduction, cellular and subcellular remodeling, and driving effector functions. Recently developed cutting‐edge imaging technologies have facilitated the study of TCR signaling and its role in actin‐dependent processes. In this review, we describe how TCR signaling cascades induce the activation of actin regulatory proteins and the formation of actin networks, and how actin dynamics is important for T‐cell homeostasis, activation, migration, and other effector functions.

Triple-Color FRET Analysis Reveals Conformational Changes in the WIP-WASp Actin-Regulating Complex

Two interaction sites provide a mechanism to finely balance the activity and degradation of an actin-regulating protein complex. WhIPping WASp into Shape Changes in the actin cytoskeleton in T cells in response to T cell receptor (TCR) activation are mediated by a complex consisting of WIP, an actin-binding protein, and WASp, a protein that promotes actin nucleation. Through a fluorescence resonance energy transfer–based study that visualized protein-protein interactions in live cells, Fried et al. revealed a two-way, end-to-end interaction between WIP and WASp. In response to TCR stimulation, one of the interactions between WIP and WASp was lost, but the other interaction remained, resulting in a conformation-induced increase in activity that stimulated actin polymerization. The conformational change also enabled the ubiquitylation and degradation of WASp to inhibit activity of the complex. Thus, the conformational change functioned as both the on and off switch. Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeletal machinery. Binding of WASp-interacting protein (WIP) to WASp modulates WASp activity and protects it from degradation. Formation of the WIP-WASp complex is crucial for the adaptive immune response. We found that WIP and WASp interacted in cells through two distinct molecular interfaces. One interaction occurred between the WASp-homology-1 (WH1) domain of WASp and the carboxyl-terminal domain of WIP that depended on the phosphorylation status of WIP, which is phosphorylated by protein kinase C θ (PKCθ) in response to T cell receptor activation. The other interaction occurred between the verprolin homology, central hydrophobic region, and acidic region (VCA) domain of WASp and the amino-terminal domain of WIP. This latter interaction required actin, because it was inhibited by latrunculin A, which sequesters actin monomers. With triple-color fluorescence resonance energy transfer (3FRET) technology, we demonstrated that the WASp activation mechanism involved dissociation of the first interaction, while leaving the second interaction intact. This conformation exposed the ubiquitylation site on WASp, leading to degradation of WASp. Together, these data suggest that the activation and degradation of WASp are delicately balanced and depend on the phosphorylation state of WIP. Our molecular analysis of the WIP-WASp interaction provides insight into the regulation of actin-dependent processes.

WASp family verprolin-homologous protein-2 (WAVE2) and Wiskott-Aldrich syndrome protein (WASp) engage in distinct downstream signaling interactions at the T cell antigen receptor site.

Background: The regulatory mechanisms and potential redundancy of the structurally related actin nucleation promoting factors, WAVE2 and WASp, are poorly understood. Results: Following T cell activation, WAVE2 and WASp are recruited to the TCR site and then dissociate. Conclusion: WAVE2 and WASp share similar recruitment mechanisms but differ in their subsequent molecular interactions and dynamics. Significance: These differences may explain their distinct functions in regulating actin-dependent processes. T cell antigen receptor (TCR) engagement has been shown to activate pathways leading to actin cytoskeletal polymerization and reorganization, which are essential for lymphocyte activation and function. Several actin regulatory proteins were implicated in regulating the actin machinery, such as members of the Wiskott-Aldrich syndrome protein (WASp) family. These include WASp and the WASp family verprolin-homologous protein-2 (WAVE2). Although WASp and WAVE2 share several structural features, the precise regulatory mechanisms and potential redundancy between them have not been fully characterized. Specifically, unlike WASp, the dynamic molecular interactions that regulate WAVE2 recruitment to the cell membrane and specifically to the TCR signaling complex are largely unknown. Here, we identify the molecular mechanism that controls the recruitment of WAVE2 in comparison with WASp. Using fluorescence resonance energy transfer (FRET) and novel triple-color FRET (3FRET) technology, we demonstrate how WAVE2 signaling complexes are dynamically regulated during lymphocyte activation in vivo. We show that, similar to WASp, WAVE2 recruitment to the TCR site depends on protein-tyrosine kinase, ZAP-70, and the adaptors LAT, SLP-76, and Nck. However, in contrast to WASp, WAVE2 leaves this signaling complex and migrates peripherally together with vinculin to the membrane leading edge. Our experiments demonstrate that WASp and WAVE2 differ in their dynamics and their associated proteins. Thus, this study reveals the differential mechanisms regulating the function of these cytoskeletal proteins.

Ubiquitylation-dependent downregulation of Nck regulates its functional activity

The Nck adapter protein is involved in key cellular functions, such as actin polymerization and reorganization, serving as a molecular bridge between the surface complex essential for foreign antigen recognition, the T‐cell antigen receptor (TCR), and the actin machinery. However, the mechanisms regulating Nck expression and functions are unknown. In this study, we revealed Nck negative regulation and demonstrated that Nck is ubiquitylated following cellular activation. We identified the molecular determinants and mediators involved in this process. Our data suggest that Nck ubiquitylation might serve as a mechanism controlling Nck‐mediated effector functions during cellular activation.

A conformational change within the WAVE2 complex regulates its degradation following cellular activation

WASp family Verprolin-homologous protein-2 (WAVE2), a member of the Wiskott-Aldrich syndrome protein (WASp) family of actin nucleation promoting factors, is a central regulator of actin cytoskeleton polymerization and dynamics. Multiple signaling pathways operate via WAVE2 to promote the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. WAVE2 exists as a part of a pentameric protein complex known as the WAVE regulatory complex (WRC), which is unstable in the absence of its individual proteins. While the involvement of WAVE2 in actin polymerization has been well documented, its negative regulation mechanism is poorly characterized to date. Here, we demonstrate that WAVE2 undergoes ubiquitylation in a T-cell activation dependent manner, followed by proteasomal degradation. The WAVE2 ubiquitylation site was mapped to lysine 45, located at the N-terminus where WAVE2 binds to the WRC. Using Förster resonance energy transfer (FRET), we reveal that the autoinhibitory conformation of the WRC maintains the stability of WAVE2 in resting cells; the release of autoinhibition following T-cell activation facilitates the exposure of WAVE2 to ubiquitylation, leading to its degradation. The dynamic conformational structures of WAVE2 during cellular activation dictate its degradation.