RESUMO
Cancer cells depend on actin cytoskeleton rearrangement to carry out hallmark malignant functions including activation, proliferation, migration and invasiveness. Wiskott-Aldrich Syndrome protein (WASp) is an actin nucleation-promoting factor and is a key regulator of actin polymerization in hematopoietic cells. The involvement of WASp in malignancies is incompletely understood. Since WASp is exclusively expressed in hematopoietic cells, we performed in silico screening to identify small molecule compounds (SMCs) that bind WASp and promote its degradation. We describe here one such identified molecule; this WASp-targeting SMC inhibits key WASp-dependent actin processes in several types of hematopoietic malignancies in vitro and in vivo without affecting naïve healthy cells. This small molecule demonstrates limited toxicity and immunogenic effects, and thus, might serve as an effective strategy to treat specific hematopoietic malignancies in a safe and precisely targeted manner.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Neoplasias Hematológicas/tratamento farmacológico , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Invasividade Neoplásica , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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 ß-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.
Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Células Matadoras Naturais/imunologia , Mecanotransdução Celular/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Actomiosina/metabolismo , Células Cultivadas , Humanos , Conformação Proteica , Transdução de Sinais/imunologiaRESUMO
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.
Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação Proteica , Família de Proteínas da Síndrome de Wiskott-Aldrich/química , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Aminoácidos/metabolismo , Linhagem Celular , Humanos , Ativação Linfocitária/imunologia , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise , Receptores de Antígenos de Linfócitos T/metabolismo , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ubiquitinação , Família de Proteínas da Síndrome de Wiskott-Aldrich/genéticaRESUMO
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.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos , Sinapses Imunológicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Transporte Proteico , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Proteínas de Transporte/química , Proteínas do Citoesqueleto/química , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Sinapses Imunológicas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteína Quinase C/metabolismo , Transdução de Sinais , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
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.
Assuntos
Actinas/química , Proteínas do Citoesqueleto/química , Transferência Ressonante de Energia de Fluorescência/métodos , Peptídeos e Proteínas de Sinalização Intracelular/química , Conformação Proteica , Proteína da Síndrome de Wiskott-Aldrich/química , Actinas/metabolismo , Sítios de Ligação/genética , Western Blotting , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/metabolismo , Células Jurkat , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutação , Fosforilação , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genética , Linfócitos T/metabolismo , Linfócitos T/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
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.
Assuntos
Etilenos/farmacologia , Integrina alfa4beta1/metabolismo , Leucemia Mieloide/tratamento farmacológico , Oxirredução/efeitos dos fármacos , Compostos de Sulfidrila/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fibronectinas/metabolismo , Células HL-60 , Humanos , Integrina alfa4beta1/antagonistas & inibidores , Leucemia Mieloide/metabolismo , Masculino , Camundongos , Camundongos SCID , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células U937 , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Protection against infectious diseases is mediated by the immune system (1,2). T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells (3,4). T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity (5). The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression (6,7). The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites (8). Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications. Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process (9,10). We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization (11). Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody (8,9), which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.
Assuntos
Microscopia Confocal/métodos , Linfócitos T/citologia , Linfócitos T/imunologia , Complexo CD3/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Processamento de Imagem Assistida por Computador , Células Jurkat , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/imunologia , TransfecçãoRESUMO
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.
Assuntos
Actinas/imunologia , Proteínas do Citoesqueleto/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Transdução de Sinais/imunologia , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Cortactina/genética , Cortactina/imunologia , Proteínas do Citoesqueleto/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Transdução de Sinais/genética , Trombocitopenia/genética , Trombocitopenia/imunologia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologiaRESUMO
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.