Maor H. Pauker

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.

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.

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.

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.

Redox Modulation of Adjacent Thiols in VLA-4 by AS101 Converts Myeloid Leukemia Cells from a Drug-Resistant to Drug-Sensitive State

Interaction between the integrin VLA-4 on acute myelogenous leukemia (AML) cells with stromal fibronectin is a decisive factor in chemotherapeutic resistance. In this study, we provide a rationale for a drug repositioning strategy to blunt integrin activation in AML cells and restore their sensitivity to chemotherapy. Specifically, we demonstrate that the nontoxic tellurium compound AS101, currently being evaluated in clinical trials, can abrogate the acquired resistance of AML. Mechanistic investigations revealed that AS101 caused redox inactivation of adjacent thiols in the exofacial domain of VLA-4 after its ligation to stromal fibronectin. This effect triggered cytoskeletal conformational changes that decreased PI3K/Akt/Bcl2 signaling, an obligatory step in chemosensitization by AS101. In a mouse xenograft of AML derived from patient leukemic cells with high VLA-4 expression and activity, we demonstrated that AS101 abrogated drug resistance and prolonged survival in mice receiving chemotherapy. Decreased integrin activity was confirmed on AML cells in vivo. The chemosensitizing activity of AS101 persisted in hosts with defective adaptive and innate immunity, consistent with evidence that integrin deactivation was not mediated by heightening immune attack. Our findings provide a mechanistic rationale to reposition the experimental clinical agent, AS101, to degrade VLA-4-mediated chemoresistance and improve clinical responses in patients with AML.

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.

Studies of novel interactions between Nck and VAV SH3 domains

Following T-cell antigen receptor (TCR) engagement, a multi-molecular complex consisting of SLP-76, Nck, and VAV1 is formed and recruited to the T-cell antigen-presenting-cell (APC) interaction site. This complex is crucial for the regulation of the actin machinery. The molecules Nck (an adaptor) and VAV1 (a GEF for small G-proteins) were previously shown to bind SLP-76. Using high-resolution imaging techniques, together with gene silencing and biochemical analysis, we studied the dynamics of this signaling complex formation. We recently showed that VAV1 and Nck can bind each other independently of SLP-76. This direct interaction is mediated by the binding of the Nck C-terminal SH3 domain and the VAV1 N-terminal SH3 domain. This interaction contributes to the cooperative nature of the complex formation. This observation was confirmed in functional studies: disruption of the Nck-VAV1 interaction strongly inhibited actin polymerization. Here, we show that Nck-VAV1 interaction is not required for Ca2+ mobilization, since a point mutation in the VAV1 N-terminal SH3 domain, which prevent the direct interaction between Nck and VAV1, has no effect on Ca2+ flux and minimal effects on ZAP-70, LAT, or PLCγ1 phosphorylation.

Dephosphorylation of the adaptor LAT and phospholipase C–γ by SHP-1 inhibits natural killer cell cytotoxicity

Researchers identify targets of a phosphatase that inhibits the activity of natural killer cells. Keeping NK cells in check Natural killer (NK) cells are immune cells that recognize and kill virally infected cells and tumor cells. Whether an NK cell becomes activated or inhibited depends on the relative extent of signaling by cell surface activating or inhibitory receptors that are engaged. Inhibitory receptor signaling activates the inhibitory phosphatase SHP-1. In experiments with human NK cells, Matalon et al. identified the adaptor protein LAT and two members of the phospholipase C–γ family as substrates of SHP-1. Inhibitory receptor signaling not only resulted in dephosphorylation of LAT, which reduced intracellular Ca2+ signaling by activating receptors, but also promoted LAT ubiquitylation and degradation, further dampening NK cell function. Natural killer (NK) cells discriminate between healthy cells and virally infected or transformed self-cells by tuning activating and inhibitory signals received through cell surface receptors. Inhibitory receptors inhibit NK cell function by recruiting and activating the tyrosine phosphatase Src homology 2 (SH2) domain–containing protein tyrosine phosphatase–1 (SHP-1) to the plasma membrane. However, to date, the guanine nucleotide exchange factor VAV1 is the only direct SHP-1 substrate identified in NK cells. We reveal that the adaptor protein linker for activation of T cells (LAT) as well as phospholipase C–γ1 (PLC-γ1) and PLC-γ2 are SHP-1 substrates. Dephosphorylation of Tyr132 in LAT by SHP-1 in NK cells abrogated the recruitment of PLC-γ1 and PLC-γ2 to the immunological synapse between the NK cell and a cancer cell target, which reduced NK cell degranulation and target cell killing. Furthermore, the ubiquitylation of LAT by the E3 ubiquitin ligases c-Cbl and Cbl-b, which was induced by LAT phosphorylation, led to the degradation of LAT in response to the engagement of inhibitory receptors on NK cells, which abrogated NK cell cytotoxicity. Knockdown of the Cbl proteins blocked LAT ubiquitylation, which promoted NK cell function. Expression of a ubiquitylation-resistant mutant LAT blocked inhibitory receptor signaling, enabling cells to become activated. Together, these data identify previously uncharacterized SHP-1 substrates and inhibitory mechanisms that determine the response of NK cells.

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.

Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation

TCR‐mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ‐chain motifs, mediating the TCRs direct interaction with actin and inducing actin bundling. While T cells expressing the ζ‐chain mutated in these motifs lack cytoskeleton (actin) associated (cska)‐TCRs, they express normal levels of non‐cska and surface TCRs as cells expressing wild‐type ζ‐chain. However, such mutant cells are unable to display activation‐dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR‐actin linkage, providing the missing gap linking between TCR‐mediated Ag recognition, specific cytoskeleton orientation toward the T‐cell–APC interacting pole and long‐lived IS maintenance.