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1.
Front Immunol ; 7: 449, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27833610

RESUMO

The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1), was chosen as a viable option, and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant, and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1-Lck complexes were detectable in primary CD4+ T-cells with their maximum levels peaking 10 s after TCR-CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394Lck, which co-purifies with high molecular weight cellular fractions. The formation of RACK1-Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1-pY394Lck-α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with nocodazole, which disrupts the microtubular network, also blocked the formation of RACK1-Lck complexes. Importantly, activation-induced Lck redistribution was diminished in primary CD4+ T-cells by an adenoviral-mediated knockdown of RACK1. These results demonstrate that in T cells, RACK1, as an essential component of the multiprotein complex which upon TCR engagement, links the binding of kinase active Lck to elements of the cytoskeletal network and affects the subcellular redistribution of Lck.

2.
Blood ; 122(9): 1634-48, 2013 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-23794064

RESUMO

In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted.


Assuntos
Análise Mutacional de DNA/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Sequenciamento de Nucleotídeos em Larga Escala , Inibidores de Proteínas Quinases/uso terapêutico , Adolescente , Adulto , Idoso , Domínio Catalítico/genética , Feminino , Proteínas de Fusão bcr-abl/química , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Estudos Retrospectivos , Adulto Jovem
3.
Immunol Lett ; 142(1-2): 64-74, 2012 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-22281390

RESUMO

Lck is the principal signal-generating tyrosine kinase of the T cell activation mechanism. We have previously demonstrated that induced Lck activation outside of lipid rafts (LR) results in the rapid translocation of a fraction of Lck to LR. While this translocation predicates the subsequent production of IL-2, the mechanism underpinning this process is unknown. Here, we describe the main attributes of this translocating pool of Lck. Using fractionation of Brij58 lysates, derived from primary naive non-activated CD4(+) T cells, we show that a significant portion of Lck is associated with high molecular weight complexes representing a special type of detergent-resistant membranes (DRMs) of relatively high density and sensitivity to laurylmaltoside, thus called heavy DRMs. TcR/CD4 coaggregation-mediated activation resulted in the redistribution of more than 50% of heavy DRM-associated Lck to LR in a microtubular network-dependent fashion. Remarkably, in non-activated CD4(+) T-cells, only heavy DRM-associated Lck is phosphorylated on its activatory tyrosine 394 and this pool of Lck is found to be membrane confined with CD45 phosphatase. These data are the first to illustrate a lipid microdomain-based mechanism concentrating the preactivated pool of cellular Lck and supporting its high stoichiometry of colocalization with CD45 in CD4(+) T cells. They also provide a new structural framework to assess the mechanism underpinning the compartmentalization of critical signaling elements and regulation of spatio-temporal delivery of Lck function during the T cell proximal signaling.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Microdomínios da Membrana/metabolismo , Transdução de Sinais , Animais , Membrana Celular/metabolismo , Centrifugação com Gradiente de Concentração , Detergentes/farmacologia , Ativação Enzimática , Feminino , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico
4.
Mol Immunol ; 48(12-13): 1439-47, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21501873

RESUMO

Death receptor-6 (DR6) apparently participates in the regulation of T-cell activation and/or activity as its genetic disruption results in enhanced CD4+ T-cell expansion, the production of Th2 cytokines, and interestingly also the compromised migration of CD4+ T cells to sites of inflammation. However, the mechanism of regulation of DR6 expression in cells of the immune system is not fully understood. In this communication we show that DR6 is not expressed in resting T cells from human peripheral blood or murine lymph nodes but that its expression is significantly upregulated in CD3 crosslinking- or PMA/ionomycin-activated T lymphocytes. DR6 expression is transiently increased in both activated human CD4+ and CD8+ T cells and it is apparently dependent on the activation of NF-κB and NF-AT signaling pathways. In contrast to primary peripheral blood T cells, the widely used model lymphoblastic leukemia T-cell line Jurkat is DR6-positive and unexpectedly, TCR-mediated stimulation of Jurkat cells strongly downregulates DR6 expression via suppression of its transcription.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Animais , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Movimento Celular , Humanos , Interleucinas/biossíntese , Células Jurkat , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Células Th2/imunologia , Células Th2/metabolismo , Regulação para Cima
6.
Transfusion ; 51(5): 1012-21, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21058954

RESUMO

BACKGROUND: Recent transmissions of variant Creutzfeldt-Jakob disease by blood transfusion emphasize the need for the development of prion screening tests. The detection of prions in blood is complicated by the presence of poorly characterized cellular prion protein (PrP(C) ) in both plasma and blood cells. According to published studies, most of PrP(C) in blood cells resides in platelets (PLTs) and white blood cells. STUDY DESIGN AND METHODS: To clarify conflicting reports about the quantity of PrP(C) associated with human red blood cells (RBCs), quantitative flow cytometry, Western blot (WB), and enzyme-linked immunosorbent assay (ELISA) were used to measure protein levels in healthy donors. RESULTS: RBCs expressed 290 ± 140 molecules of PrP(C) per cell, assuming equimolar binding of monoclonal antibody (MoAb) 6H4 to PrP(C). Binding of alternate PrP(C) MoAbs, FH11 and 3F4, was substantially lower. WB estimated the level of PrP(C) per cell on RBCs to be just four times lower than in PLTs. A similar level of PrP(C) was detected using ELISA. The weak binding of commonly used MoAb 3F4 was not caused by PrP(C) conformation, truncation, or glycosylation, suggesting a covalent modification, likely glycation, of the 3F4 epitope. CONCLUSIONS: Taken together, human RBCs express low but significant amounts of PrP(C) /cell, which makes them, due to high RBC numbers, major contributors to the pool of cell-associated PrP(C) in blood. Previous reports utilizing MoAb 3F4 may have underestimated the amount of PrP(C) in RBCs. Likewise, screening tests for the presence of the abnormal prion protein in blood may be difficult if the abnormal protein is modified similar to RBC PrP(C).


Assuntos
Síndrome de Creutzfeldt-Jakob , Eritrócitos/metabolismo , Proteínas PrPC/metabolismo , Reação Transfusional , Anticorpos Monoclonais/imunologia , Western Blotting , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/prevenção & controle , Síndrome de Creutzfeldt-Jakob/transmissão , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Membrana Eritrocítica/metabolismo , Citometria de Fluxo , Glicosilação , Humanos , Programas de Rastreamento/métodos , Proteínas PrPC/imunologia
7.
Thromb Haemost ; 102(5): 966-74, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19888536

RESUMO

The recently shown transmissibility of variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion emphasises the need for better understanding of the cellular prion protein (PrPc) in blood. A substantial amount of cell-associated PrPc in blood resides in platelets. Platelet activation leads to up-regulation of PrPc on the platelet surface and its release on exosomes and microparticles. The sub-cellular localisation and function of platelet PrPc, however, is poorly understood. In the present study, we investigated the association of PrPc with platelet lipid rafts and the platelet cytoskeleton. Immuno-fluorescence microscopy showed that the signals of PrPc and P-selectin, both of which occupy intracellular alpha granules, were separated on the membrane, suggesting organisation in different membrane domains. A flotation assay of platelet lysates demonstrated that a relatively small portion of platelet PrPc floats with lipid rafts, regardless of platelet activation status. This was reversed by depolymerisation of the platelet cytoskeleton, which led to flotation of most platelet PrPc, suggesting that interactions with the cytoskeleton prevent flotation of PrPc rafts. This association of PrPc with the platelet cytoskeleton was confirmed by its presence in both the isolated membrane skeleton and actin cytoskeleton. Platelet activation significantly increased the amount of PrPc associated with the cytoskeleton. Our results indicate that the localisation of PrPc in platelets is complex, with the majority of PrPc present within platelet lipid rafts linked to the platelet cytoskeleton. This localisation places PrPc in a position where it can interact with proteins involved in platelet signalling and eventually with vCJD prions.


Assuntos
Plaquetas/química , Citoesqueleto/química , Microdomínios da Membrana/química , Proteínas PrPC/sangue , Biopolímeros , Plaquetas/ultraestrutura , Membrana Celular/química , Grânulos Citoplasmáticos/química , Humanos , Microscopia de Fluorescência , Selectina-P , Ativação Plaquetária
8.
J Biomed Mater Res A ; 86(3): 769-78, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18041724

RESUMO

Using the layer-by-layer technique, ELISA polystyrene plates were coated with multilayer assemblies of albumin with various heparins or with multilayer assemblies of albumin. The coatings containing heparin were tested for their ability to potentiate thrombin inhibition by antithrombin and its dependence on the layer arrangement. The order of activities of surface bound heparins matched their order in solution; however their activity was reduced to less than 10% due to binding. The increasing number of layers increased the activity of the coatings suggesting that heparin inside the assemblies is available for the interaction. The albumin-heparin assemblies overcoated with albumin layers preserved about half of heparin activity. Platelets adhered in similar amounts to albumin-heparin and albumin coatings; however, in both cases platelets adhered more to single layer than to multilayer coatings. The adhesion of platelets to single layer coatings was also affected by the crosslinking of the coatings; more platelets adhered to less crosslinked single layer coatings while multilayer coatings remained essentially unaffected by crosslinking. If the coatings were dried and reswollen, a substantial number of platelets adhered to the reconditioned single layer coatings but the two layer coatings were affected much less and the adhesion of platelets to the coatings with three layers was close to normal. A minimum of three albumin-heparin or albumin layers is apparently required to shield the underlying surface and to achieve proper functioning of the coatings.


Assuntos
Materiais Revestidos Biocompatíveis/síntese química , Heparina/metabolismo , Teste de Materiais/métodos , Adesividade Plaquetária , Soroalbumina Bovina/metabolismo , Trombina/antagonistas & inibidores , Animais , Bovinos , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Adesividade Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Espectroscopia de Infravermelho com Transformada de Fourier , Suínos
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