RESUMO
Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling.
Assuntos
Efrina-A5/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 12/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Células COS , Caspase 3/metabolismo , Membrana Celular/metabolismo , Chlorocebus aethiops , Citoesqueleto/metabolismo , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Receptor EphA3RESUMO
Activated Ras has been found in many types of cancer. However, the mechanism underlying Ras-promoted tumor metastasis remains unclear. We demonstrate here that activated Ras induces tyrosine dephosphorylation and inhibition of FAK mediated by the Ras downstream Fgd1-Cdc42-PAK1-MEK-ERK signaling cascade. ERK phosphorylates FAK S910 and recruits PIN1 and PTP-PEST, which colocalize with FAK at the lamellipodia of migrating cells. PIN1 binding and prolyl isomerization of FAK cause PTP-PEST to interact with and dephosphorylate FAK Y397. Inhibition of FAK mediated by this signal relay promotes Ras-induced cell migration, invasion, and metastasis. These findings uncover the importance of sequential modification of FAK-by serine phosphorylation, isomerization, and tyrosine dephosphorylation--in the regulation of FAK activity and, thereby, in Ras-related tumor metastasis.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 12/metabolismo , Proteínas ras/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Immunoblotting , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/genética , Células NIH 3T3 , Peptidilprolil Isomerase de Interação com NIMA , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Peptidilprolil Isomerase/genética , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 12/genética , Serina/metabolismo , Transfecção , Tirosina/metabolismo , Proteínas ras/genéticaRESUMO
Alzheimer's disease (AD) is the most common cause of dementia worldwide. The pathogenesis of this neurodegenerative disease, currently without curative treatment, is associated with the accumulation of amyloid ß (Aß) in brain parenchyma and cerebral vasculature. AD patients are unable to clear this toxic peptide, leading to Aß accumulation in their brains and, presumably, the pathology associated with this devastating disease. Compounds that stimulate the immune system to clear Aß may therefore have great therapeutic potential in AD patients. Monophosphoryl lipid A (MPL) is an LPS-derived Toll-like receptor 4 agonist that exhibits unique immunomodulatory properties at doses that are nonpyrogenic. We show here that repeated systemic injections of MPL, but not LPS, significantly improved AD-related pathology in APP(swe)/PS1 mice. MPL treatment led to a significant reduction in Aß load in the brain of these mice, as well as enhanced cognitive function. MPL induced a potent phagocytic response by microglia while triggering a moderate inflammatory reaction. Our data suggest that the Toll-like receptor 4 agonist MPL may be a treatment for AD.
Assuntos
Doença de Alzheimer/prevenção & controle , Encéfalo/efeitos dos fármacos , Lipídeo A/análogos & derivados , Receptor 4 Toll-Like/agonistas , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Imunidade Inata/efeitos dos fármacos , Ligantes , Lipídeo A/administração & dosagem , Lipídeo A/uso terapêutico , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microscopia de Fluorescência , Fagocitose/efeitos dos fármacos , Presenilina-1/genética , Presenilina-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 4 Toll-Like/metabolismoRESUMO
PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.
Assuntos
Movimento Celular/fisiologia , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 12/metabolismo , Substituição de Aminoácidos , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos Knockout , Mutação de Sentido Incorreto , Proteína Tirosina Fosfatase não Receptora Tipo 12/genética , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de srcRESUMO
The protein tyrosine phosphatase PEST (PTP-PEST) is involved in the regulation of the actin cytoskeleton. Despite the emerging functions attributed to both PTPs and the actin cytoskeleton in apoptosis, the involvement of PTP-PEST in apoptotic cell death remains to be established. Using several cell-based assays, we showed that PTP-PEST participates in the regulation of apoptosis. As apoptosis progressed, a pool of PTP-PEST localized to the edge of retracting lamellipodia. Expression of PTP-PEST also sensitized cells to receptor-mediated apoptosis. Concertedly, specific degradation of PTP-PEST was observed during apoptosis. Pharmacological inhibitors, immunodepletion experiments, and in vitro cleavage assays identified caspase-3 as the primary regulator of PTP-PEST processing during apoptosis. Caspase-3 specifically cleaved PTP-PEST at the (549)DSPD motif and generated fragments, some of which displayed increased catalytic activity. Moreover, caspase-3 regulated PTP-PEST interactions with paxillin, leupaxin, Shc, and PSTPIP. PTP-PEST acted as a scaffolding molecule connecting PSTPIP to additional partners: paxillin, Shc, Csk, and activation of caspase-3 correlated with the modulation of the PTP-PEST adaptor function. In addition, cleavage of PTP-PEST facilitated cellular detachment during apoptosis. Together, our data demonstrate that PTP-PEST actively contributes to the cellular apoptotic response and reveal the importance of caspases as regulators of PTPs in apoptosis.
Assuntos
Apoptose , Caspase 3/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Caspase 3/farmacologia , Catálise/efeitos dos fármacos , Extensões da Superfície Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 12 , Proteínas Tirosina Fosfatases/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato/efeitos dos fármacosRESUMO
Our inability to effectively "drug" targets such as MYC for therapeutic purposes requires the development of new approaches. We report on the implementation of a phenotype-based assay for monitoring MYC expression in multiple myeloma cells. The open reading frame (ORF) encoding an unstable variant of GFP was engineered immediately downstream of the MYC ORF using CRISPR/Cas9, resulting in co-expression of both proteins from the endogenous MYC locus. Using fluorescence readout as a surrogate for MYC expression, we implemented a pilot screen in which â¼10,000 compounds were prosecuted. Among known MYC expression inhibitors, we identified cardiac glycosides and cytoskeletal disruptors to be quite potent. We demonstrate the power of CRISPR/Cas9 engineering in establishing phenotype-based assays to identify gene expression modulators.
Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Bufanolídeos/farmacologia , Sistemas CRISPR-Cas/genética , Glicosídeos Cardíacos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Antibody-mediated capture of amyloid-beta (Aß) in peripheral blood was identified as an attractive strategy to eliminate cerebral toxic amyloid in Alzheimer's disease (AD) patients and murine models. Alternatively, defective capacity of peripheral monocytes to engulf Aß was reported in individuals with AD. In this report, we developed different approaches to investigate cellular uptake and phagocytosis of Aß, and to examine how two immunological devices--an immunostimulatory Adjuvant System and different amyloid specific antibodies--may affect these biological events. Between one and thirteen months of age, APPswe X PS1.M146V (TASTPM) AD model mice had decreasing concentrations of Aß in their plasma. In contrast, the proportion of blood monocytes containing Aß tended to increase with age. Importantly, the TLR-agonist containing Adjuvant System AS01B primed monocytes to promote de novo Aß uptake capacity, particularly in the presence of anti-Aß antibodies. Biochemical experiments demonstrated that cells achieved Aß uptake and internalization followed by Aß degradation via mechanisms that required effective actin polymerization and proteolytic enzymes such as insulin-degrading enzyme. We further demonstrated that both Aß-specific monoclonal antibodies and plasma from Aß-immunized mice enhanced the phagocytosis of 1 µm Aß-coated particles. Together, our data highlight a new biomarker testing to follow amyloid clearance within the blood and a mechanism of Aß uptake by peripheral monocytes in the context of active or passive immunization, and emphasize on novel approaches to investigate this phenomenon.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Fagocitose/imunologia , Actinas/metabolismo , Adjuvantes Imunológicos , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Combinação de Medicamentos , Imunofenotipagem , Imunoterapia , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Camundongos , Camundongos Transgênicos , Multimerização Proteica , Proteólise , Saponinas/administração & dosagem , Saponinas/imunologia , VacinaçãoRESUMO
With more than 12 million people affected worldwide, 2 million new cases occurring per year, and the rapid emergence of drug resistance and treatment failure, leishmaniasis is an infectious disease for which research on drug and vaccine development, host-pathogen, and vector-parasite interactions are current international priorities. Upon Leishmania-macrophage interaction, activation of the protein tyrosine phosphatase (PTP) SHP-1 rapidly leads to the down-regulation of Janus kinase and mitogen-activated protein kinase signaling, resulting in the attenuation of host innate inflammatory responses and of various microbicidal macrophage functions. We report that, in addition to SHP-1, the PTPs PTP1B and TCPTP are activated and posttranslationally modified in infected macrophages, and we identify an essential role for PTP1B in the in vivo progression of Leishmania infection. The mechanism underlying PTP modulation involves the proteolytic activity of the Leishmania surface protease GP63. Access of GP63 to macrophage PTP1B, TCPTP, and SHP-1 is mediated in part by a lipid raft-dependent mechanism, resulting in PTP cleavage and stimulation of phosphatase activity. Collectively, our data present a mechanism of cleavage-dependent activation of macrophage PTPs by an obligate intracellular pathogen and show that internalization of GP63, a key Leishmania virulence factor, into host macrophages is a strategy the parasite uses to interact and survive within its host.
Assuntos
Leishmania major/fisiologia , Leishmania major/patogenicidade , Metaloendopeptidases/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Proteínas de Protozoários/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Interações Hospedeiro-Parasita/fisiologia , Humanos , Leishmania major/genética , Leishmaniose/parasitologia , Leishmaniose/fisiopatologia , Macrófagos/parasitologia , Macrófagos/fisiologia , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologiaRESUMO
The Leishmania parasite is a widespread disease threat in tropical areas, causing symptoms ranging from skin lesions to death. Leishmania parasites typically invade macrophages but are also capable of infecting fibroblasts, which may serve as a reservoir for recurrent infection. Invasion by intracellular pathogens often involves exploitation of the host cell cytoskeletal and signaling machinery. Here we have observed a dramatic rearrangement of the actin cytoskeleton and marked modifications in the profile of protein tyrosine phosphorylation in fibroblasts infected with Leishmania major. Correspondingly, exposure to L. major resulted in degradation of the phosphorylated adaptor protein p130Cas and the protein-tyrosine phosphatase-PEST. Cellular and in vitro assays using pharmacological protease inhibitors, recombinant enzyme, and genetically modified strains of L. major identified the parasite protease GP63 as the principal catalyst of proteolysis during infection. A number of additional signaling proteins were screened for degradation during L. major infection as follows: a small subset was cleaved, including cortactin, T-cell protein-tyrosine phosphatase, and caspase-3, but the majority remained unaffected. Protein degradation occurred in cells incubated with Leishmania extracts in the absence of intact parasites, suggesting a mechanism permitting transfer of functional GP63 into the intracellular space. Finally, we evaluated the impact of Leishmania on MAPK signaling; unlike p44/42 and JNK, p38 was inactivated upon infection in a GP63- and protein degradation-dependent manner, which likely involves cleavage of the upstream adaptor TAB1. Our results establish that GP63 plays a central role in a number of hostcell molecular events that likely contribute to the infectivity of Leishmania.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Leishmania major/enzimologia , Leishmaniose Cutânea/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloendopeptidases/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Caspase 3/metabolismo , Cortactina/metabolismo , Proteína Substrato Associada a Crk , Leishmania major/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismoRESUMO
Apoptosis is a precisely controlled physiological mechanism that is required for the elimination of cells during embryonic development, in response to stress and infection as well as in the maintenance of homeostasis. Since the outcome of several of these biological processes is regulated by signaling events involving tyrosine phosphorylation, members of the protein tyrosine phosphatase (PTP) gene family are expected to be of primary importance. Here, we summarize the current literature linking the activities of classical PTPs with the regulation of apoptosis. The recent discovery of caspase-cleavage mediated modulation of a member of this family, PTP-PEST, indicates that other PTPs could be modulated in a similar manner. In light of this, we present an analysis of all murine and human PTPs gene for the presence of putative caspase cleavage motifs. Additional studies linking the activity of PTPs to their own regulation during programmed cell death initiation should provide important insight into the understanding of this fundamental physiological phenomenon.
Assuntos
Apoptose/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Animais , Sobrevivência Celular/fisiologia , Humanos , Transdução de Sinais/fisiologiaRESUMO
The tyrosine phosphatase PTP-PEST has been implicated in the regulation of cell spreading and migration through dephosphorylation of focal adhesion proteins and inhibition of Rac GTPase activity. The focal adhesion adaptor protein paxillin is also necessary for normal cell migration and binds directly to PTP-PEST. In this study, we have utilized PTP-PEST(-/-) and paxillin(-/-) fibroblasts to demonstrate that paxillin is essential for PTP-PEST inhibition of cell spreading and membrane protrusion as well as inhibition of adhesion-induced Rac activation. Furthermore, we show that paxillin-binding is necessary for PTP-PEST stimulation of cell migration. Mutation analysis indicates that PTP-PEST function involves binding to the paxillin C-terminal LIM domains, and signaling through the tyrosine 31 and 118 phosphorylation sites, as well as the LD4 motif of the paxillin N-terminus. Using 'substrate trapping' approaches and immunoprecipitation, we show that the ARF GAP paxillin kinase linker PKL/GIT2, a paxillin LD4 binding partner, is a substrate for PTP-PEST. Additionally, the PKL-paxillin interaction was necessary for PTP-PEST inhibition of cell spreading. These data provide mechanistic insight into how the paxillin-PTP-PEST interaction contributes to integrin signaling events associated with the spatiotemporal regulation of key modulators of the cytoskeleton and cell motility machinery.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Motivos de Aminoácidos/genética , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Citoesqueleto/genética , Fibroblastos/citologia , Proteínas Ativadoras de GTPase , Integrinas/genética , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Paxilina/genética , Paxilina/metabolismo , Fosfoproteínas/genética , Proteína Tirosina Fosfatase não Receptora Tipo 12 , Proteínas Tirosina Fosfatases/deficiênciaRESUMO
PSTPIP is a tyrosine-phosphorylated protein involved in the organization of the cytoskeleton. Its ectopic expression induces filipodial-like membrane extensions in NIH 3T3 cells. We previously observed a defect in cytokinesis and an increase in the tyrosine phosphorylation of PSTPIP in PTP-PEST-deficient fibroblasts. In this article, we demonstrate that PTP-PEST and PSTPIP are found in the same complexes in vivo and that they interact directly through the CTH domain of PTP-PEST and the coiled-coil domain of PSTPIP. We tested pathways that could regulate the tyrosine phosphorylation of PSTPIP. We found that the activation of the epidermal growth factor and platelet-derived growth factor receptors can induce PSTPIP phosphorylation. With the use of the PP2 inhibitor, we demonstrate that Src kinases are not involved in the epidermal growth factor-mediated phosphorylation of PSTPIP. Together with previous results, this suggests that c-Abl is the critical tyrosine kinase downstream of growth factor receptors responsible for PSTPIP phosphorylation. We also demonstrate that PTP-PEST dephosphorylates PSTPIP at tyrosine 344. Importantly, we identified tyrosine 344 as the main phosphorylation site of PSTPIP by performing tryptic phosphopeptide maps. This is an important finding since tyrosine 367 of PSTPIP was also proposed as a candidate phosphorylation site involved in the negative regulation of the association between PSTPIP and WASP. In this respect, we observed that the PSTPIP.WASP complex is stable in vivo and is not affected by the phosphorylation of PSTPIP. Furthermore, we demonstrate that PSTPIP serves as a scaffold protein between PTP-PEST and WASP and allows PTP-PEST to dephosphorylate WASP. This finding suggests a possible mechanism for PTP-PEST to directly modulate actin remodeling through the PSTPIP-WASP interaction.