Omri Matalon

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.

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.

WIP: more than a WASp-interacting protein

WIP plays an important role in the remodeling of the actin cytoskeleton, which controls cellular activation, proliferation, and function. WIP regulates actin polymerization by linking the actin machinery to signaling cascades. WIP binding to WASp and to its homolog, N‐WASp, which are central activators of the actin‐nucleating complex Arp2/3, regulates their cellular distribution, function, and stability. By binding to WASp, WIP protects it from degradation and thus, is crucial for WASp retention. Indeed, most mutations that result in WAS, an X‐linked immunodeficiency caused by defective/absent WASp activity, are located in the WIP‐binding region of WASp. In addition, by binding directly to actin, WIP promotes the formation and stabilization of actin filaments. WASp‐independent activities of WIP constitute a new research frontier and are discussed extensively in this article. Here, we review the current information on WIP in human and mouse systems, focusing on its associated proteins, its molecular‐regulatory mechanisms, and its role as a key regulator of actin‐based processes in the immune system.

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.

WIP Remodeling Actin behind the Scenes: How WIP Reshapes Immune and Other Functions

Actin polymerization is a fundamental cellular process regulating immune cell functions and the immune response. The Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor, which is exclusively expressed in hematopoietic cells, where it plays a key regulatory role in cytoskeletal dynamics. WASp interacting protein (WIP) was first discovered as the binding partner of WASp, through the use of the yeast two hybrid system. WIP was later identified as a chaperone of WASp, necessary for its stability. Mutations occurring at the WASp homology 1 domain (WH1), which serves as the WIP binding site, were found to cause the Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). WAS manifests as an immune deficiency characterized by eczema, thrombocytopenia, recurrent infections, and hematopoietic malignancies, demonstrating the importance of WIP for WASp complex formation and for a proper immune response. WIP deficiency was found to lead to different abnormalities in the activity of various lymphocytes, suggesting differential cell-dependent roles for WIP. Additionally, WIP deficiency causes cellular abnormalities not found in WASp-deficient cells, indicating that WIP fulfills roles beyond stabilizing WASp. Indeed, WIP was shown to interact with various binding partners, including the signaling proteins Nck, CrkL and cortactin. Recent studies have demonstrated that WIP also takes part in non immune cellular processes such as cancer invasion and metastasis, in addition to cell subversion by intracellular pathogens. Understanding of numerous functions of WIP can enhance our current understanding of activation and function of immune and other cell types.

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.

Actin retrograde flow controls natural killer cell response by regulating the conformation state of SHP‐1

Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin–myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between β‐actin and the SH2‐domain‐containing protein tyrosine phosphatase‐1 (SHP‐1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP‐1 in additional cellular systems.

The Ebola-Glycoprotein Modulates the Function of Natural Killer Cells

The Ebola virus (EBOV) uses evasion mechanisms that directly interfere with host T-cell antiviral responses. By steric shielding of human leukocyte antigen class-1, the Ebola glycoprotein (GP) blocks interaction with T-cell receptors (TCRs), thus rendering T cells unable to attack virus-infected cells. It is likely that this mechanism could promote increased natural killer (NK) cell activity against GP-expressing cells by preventing the engagement of NK inhibitory receptors; however, we found that primary human NK cells were less reactive to GP-expressing HEK293T cells. This was manifested as reduced cytokine secretion, a reduction in NK degranulation, and decreased lysis of GP-expressing target cells. We also demonstrated reduced recognition of GP-expressing cells by recombinant NKG2D and NKp30 receptors. In accordance, we showed a reduced monoclonal antibody-based staining of NKG2D and NKp30 ligands on GP-expressing target cells. Trypsin digestion of the membrane-associated GP led to a recovery of the recognition of membrane-associated NKG2D and NKp30 ligands. We further showed that membrane-associated GP did not shield recognition by KIR2DL receptors; in accordance, GP expression by target cells significantly perturbed signal transduction through activating, but not through inhibitory, receptors. Our results suggest a novel evasion mechanism employed by the EBOV to specifically avoid the NK cell immune response.

Cbl ubiquitin ligases mediate the inhibition of natural killer cell activity

ABSTRACT Natural killer (NK) cells are essential for killing transformed and virally infected cells. To prevent auto-reactivity, NK cell activation is inhibited by inhibitory receptors that activate the tyrosine phosphatase SHP-1, which dephosphorylates signaling molecules crucial for NK cell activation. Initially, only a single SHP-1 substrate was identified in NK cells, the GEF VAV1. We recently demonstrated that under inhibitory conditions, LAT, PLCγ1 and PLCγ2 serve as novel SHP-1 substrates in NK cells. Furthermore, we showed that during NK cell inhibition, LAT is ubiquitylated by c-Cbl and Cbl-b, leading to its proteasomal degradation, abolishing NK cell cytotoxicity. Here, we address the mechanism through which the Cbl proteins are activated following inhibitory receptor engagement. We demonstrate that during NK cell inhibition, the expression level of the Cbl proteins significantly increases. These data suggest that inhibitory KIR receptors regulate the stability of the Cbl proteins, thereby enabling Cbl-mediated inhibition of NK cell cytotoxicity.

WASp and WAVE Proteins: From Structure, Through Function, to Clinical Aspects

T cells play a pivotal role in adoptive immunity, both in cell mediated cytotoxicity and in the activation of the humoral immune response. In order to perform their effector function, T cells undergo dramatic morphological changes upon activation. These changes enable their binding to and extravasation through the vascular endothelium into the neighboring tissue, the formation of an immunological synapse (IS) with an antigen-presenting cell (APC), and subsequently, the polarized secretion of cytokines and/or cytolytic granules, leading to the execution of effector functions. The actin cytoskeleton is directly involved in all these processes. Thus, it is crucial for T cell mediated immune responses, providing a dynamic and flexible platform for signal transduction, cellular and subcellular remodeling, and for driving effector functions. The actin-regulatory proteins, Wiskott-Aldrich syndrome protein (WASp) and WASp family Verprolin-homologous protein (WAVE) play key roles in T cell biology. In this review, we will focus on these two proteins, describing their structure, recruitment, activation and function. Finally, we will address pathological aspects related to defects in these actin regulators.