Transmembránové adaptorové proteiny nemají enzymatickou nebo kinázovou funkci, ale jsou zapojeny do přenosu signálu z buněčného povrchu do buněčného jádra. Na zvířecím modelu vede změna exprese adaptorové molekuly PAG k obrazu leukemie. V této studii byly vyšetřovány adaptorové molekuly PAG, LAT a NTAL ve fyziologických lymfocytárních prekurzorech a na souboru 75 diagnostických vzorků dětské akutní lymfoblastické leukemie (ALL). Byla analyzována exprese těchto molekul na mRNA úrovni (metodou kvantitativní reverzně-transkriptázové polymerázové řetězové reakce) a na úrovni proteinu (pomocí průtokové cytometrie). V průběhu fyziologické lymfocytární maturace se hladina některých adaptorů signifikantně mění mezi jednotlivými stadii. V různých podtypech ALL je možno sledovat odlišnou dynamiku exprese jednotlivých molekul, zejména TEL/AML1 pozitivní pacienti jsou charakterizováni unikátním profilem exprese. Hladina NTAL při diagnóze T-ALL je prognosticky významným faktorem predikujícím odpověď na iniciální léčbu kortikosteroidy. Tento jev se podařilo potvrdit i v in vitro experimentu – při inkubaci s kortikoidy je procento přežívajících buněk vyšší u nativní linie T-buněčné leukemie než u stejné linie s transfekovaným genem NTAL.

Transmembrane adaptor proteins do not posses an enzymatic or kinase function, but they mediate signal transmission from cell surface to the nucleus. In animal model, changes in PAG adaptor molecule expression induce a leukaemia-like phenotype. In this study, adaptor molecules PAG, LAT, NTAL were analysed in physiological lymphocyte precursors and in 75 diagnostic samples of childhood acute lymphoblastic leukaemia (ALL). The expression status was assessed at mRNA level (using real-time quantitative reverse-transcriptase polymerase chain reaction) and at protein level (using flow cytometry). During a physiological maturation of lymphocyte precursors an expression status of some adaptors shows significant dynamics among distinct maturation stages. Among different ALL subgroups different adaptor molecules expression dynamics can be seen, particularly in TEL/AML1 patients displaying a unique profile of expression. NTAL level at the diagnosis of T-ALL has a prognostic impact predicting good or poor response to initial corticosteroid treatment. This observation was confirmed in in vitro assay – after incubation with corticoids a percentage of surviving cells is higher in wild-type T-ALL cell line compared to the same cells with transfected NTAL gene.

• MeSH
• adaptorové proteiny signální transdukční MeSH
• akutní lymfatická leukemie genetika   imunologie   MeSH
• B-lymfocyty MeSH
• financování organizované MeSH
• Jurkat buňky cytologie   MeSH
• messenger RNA MeSH
• polymerázová řetězová reakce využití   MeSH
• prekurzory T-buněk MeSH
• průtoková cytometrie využití   MeSH
• regulace genové exprese u leukemie MeSH
• T-lymfocyty MeSH
• techniky in vitro MeSH

Chemotaxis of mast cells is one of the crucial steps in their development and function. Non-T cell activation linker (NTAL) is a transmembrane adaptor protein that inhibits the activation of mast cells and B cells in a phosphorylation-dependent manner. Here, we studied the role of NTAL in the migration of mouse mast cells stimulated by prostaglandin E2 (PGE2). Although PGE2 does not induce the tyrosine phosphorylation of NTAL, unlike IgE immune complex antigens, we found that loss of NTAL increased the chemotaxis of mast cells toward PGE2 Stimulation of mast cells that lacked NTAL with PGE2 enhanced the phosphorylation of AKT and the production of phosphatidylinositol 3,4,5-trisphosphate. In resting NTAL-deficient mast cells, phosphorylation of an inhibitory threonine in ERM family proteins accompanied increased activation of β1-containing integrins, which are features often associated with increased invasiveness in tumors. Rescue experiments indicated that only full-length, wild-type NTAL restored the chemotaxis of NTAL-deficient cells toward PGE2 Together, these data suggest that NTAL is a key inhibitor of mast cell chemotaxis toward PGE2, which may act through the RHOA/ERM/β1-integrin and PI3K/AKT axes.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• aktiny metabolismus   MeSH
• antigeny CD29 metabolismus   MeSH
• bodová mutace MeSH
• chemotaxe * MeSH
• cholesterol metabolismus   MeSH
• dinoproston metabolismus   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosforylace MeSH
• integriny metabolismus   MeSH
• mastocyty metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• myši MeSH
• proteinové domény MeSH
• proteiny metabolismus   MeSH
• signální transdukce MeSH
• threonin chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The transmembrane adaptor protein NTAL (non-T-cell activation linker) participates in signalosome assembly in hematopoietic cells, but its exact role in cell physiology remains enigmatic. We report here that BM-derived mast cells from NTAL-deficient mice, responding to Ag alone or in combination with SCF, exhibit reduced spreading on fibronectin, enhanced filamentous actin depolymerization and enhanced migration towards Ag relative to WT cells. No such differences between WT and NTAL(-/-) BM-derived mast cells were observed when SCF alone was used as activator. We have examined the activities of two small GTPases, Rac and Rho, which are important regulators of actin polymerization. Stimulation with Ag and/or SCF enhanced activity of Rac(1,2,3) in both NTAL(-/-) and WT cells. In contrast, RhoA activity decreased and this trend was much faster and more extensive in NTAL(-/-) cells, indicating a positive regulatory role of NTAL in the recovery of RhoA activity. After restoring NTAL into NTAL(-/-) cells, both spreading and actin responses were rescued. This is the first report of a crucial role of NTAL in signaling, via RhoA, to mast cell cytoskeleton.

• MeSH
• aktiny genetika   imunologie   metabolismus   MeSH
• antigeny imunologie   MeSH
• buňky kostní dřeně cytologie   imunologie   metabolismus   MeSH
• cytoskelet genetika   imunologie   metabolismus   MeSH
• mastocyty cytologie   imunologie   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• proteiny genetika   imunologie   metabolismus   MeSH
• rac proteiny vázající GTP genetika   imunologie   metabolismus   MeSH
• rho proteiny vázající GTP genetika   imunologie   metabolismus   MeSH
• signální transdukce genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The biology of T-cell acute lymphoblastic leukemia (ALL) is characterized by functional pre-T-cell receptor (TCR) signaling. Non-T-cell activation linker (NTAL) is a nonenzymatic transmembrane adaptor molecule that is involved in the proximal signaling of lymphocytes. In our previous work, we found an association between high NTAL expression in T-cell ALL blasts and a favorable response to initial glucocorticoid treatment. In the present study, we confirm our previous observation in an experimental model. In addition, the molecular mechanism of the contribution of NTAL to malignant T-cell ALL blast signaling and to methylprednisolone-induced cell death is analyzed. In the in vitro experiments, we used the T-cell ALL Jurkat cell line (Jurkat/wt) and derived Jurkat cell line with stable NTAL expression (Jurkat/NTAL(+)). Cell signaling and cell death after methylprednisolone treatment and after TCR stimulation were analyzed using flow cytometry, Western blot, and quantitative polymerase chain reaction. Jurkat/NTAL(+) cells are significantly more sensitive to both methylprednisolone treatment and TCR-induced stimulation. In addition, after TCR stimulation, Jurkat/NTAL(+) cells show a higher level of intracellular extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and increased expression of the CD69 activation marker on the cell surface than the Jurkat/wt cells. The ERK inhibitor U0126 almost completely abrogates TCR-induced cell death and, importantly, reverses the sensitizing effect of the NTAL protein on methylprednisolone-induced cell death. In conclusion, NTAL acts as a tumor suppressor that enhances the proximal signaling of leukemic blasts. The key downstream molecule responsible for the biological effect of TCR signaling is ERK. Higher ERK phosphorylation leads to enhanced cell death after TCR stimulation and increases cell sensitivity to methylprednisolone-induced cell death.

• MeSH
• adaptorové proteiny signální transdukční fyziologie   MeSH
• apoptóza účinky léků   fyziologie   MeSH
• butadieny farmakologie   MeSH
• CD antigeny metabolismus   MeSH
• chemorezistence účinky léků   MeSH
• diferenciační antigeny T-lymfocytů metabolismus   MeSH
• fosforylace účinky léků   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• Jurkat buňky účinky léků   enzymologie   MeSH
• lektiny typu C metabolismus   MeSH
• lidé MeSH
• lymfoblastická leukemie-lymfom z prekurzorových T-buněk metabolismus   patologie   MeSH
• MAP kinasový signální systém účinky léků   MeSH
• methylprednisolon farmakologie   MeSH
• mitogenem aktivovaná proteinkinasa 1 antagonisté a inhibitory   metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 3 antagonisté a inhibitory   metabolismus   MeSH
• nádorové proteiny antagonisté a inhibitory   fyziologie   MeSH
• nitrily farmakologie   MeSH
• posttranslační úpravy proteinů účinky léků   MeSH
• receptory antigenů T-buněk účinky léků   MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO) and wild type mice suggested that NTAL is a negative regulator of FcεRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD) in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD) have different physiological consequences, we compared under well defined conditions FcεRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs) with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FcεRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.

• MeSH
• adaptorové proteiny vezikulární transportní genetika   metabolismus   MeSH
• genetická transkripce fyziologie   MeSH
• mastocyty metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• receptory IgE metabolismus   MeSH
• signální transdukce imunologie   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca(2+) response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)(2) or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcεRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)(2) fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcεRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• antigeny CD9 fyziologie   MeSH
• antigeny CD98 - lehké řetězce metabolismus   MeSH
• antigeny metabolismus   MeSH
• biologické modely MeSH
• buněčná membrána metabolismus   MeSH
• chemotaxe MeSH
• cytoskelet metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• fosforylace MeSH
• glukuronidasa metabolismus   MeSH
• imunoglobuliny - Fab fragmenty chemie   MeSH
• krysa rodu rattus MeSH
• mastocyty cytologie   MeSH
• membránové proteiny metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• potkani Wistar MeSH
• receptory IgE metabolismus   MeSH
• transportní systém aminokyselin y+ metabolismus   MeSH
• tyrosin chemie   MeSH
• vápník metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakty MeSH

• MeSH
• fosfoproteiny genetika   chemie   metabolismus   MeSH
• lidé MeSH
• lymfoidní tkáň metabolismus   MeSH
• membránové proteiny genetika   izolace a purifikace   metabolismus   MeSH
• receptory antigenů B-buněk metabolismus   MeSH
• receptory Fc metabolismus   MeSH
• transdukce genetická MeSH
• transportní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

Engagement of the FcepsilonRI in mast cells and basophils leads to a rapid tyrosine phosphorylation of the transmembrane adaptors LAT (linker for activation of T cells) and NTAL (non-T cell activation linker, also called LAB or LAT2). NTAL regulates activation of mast cells by a mechanism, which is incompletely understood. Here we report properties of rat basophilic leukemia cells with enhanced or reduced NTAL expression. Overexpression of NTAL led to changes in cell morphology, enhanced formation of actin filaments and inhibition of the FcepsilonRI-induced tyrosine phosphorylation of the FcepsilonRI subunits, Syk kinase and LAT and all downstream activation events, including calcium and secretory responses. In contrast, reduced expression of NTAL had little effect on early FcepsilonRI-induced signaling events but inhibited calcium mobilization and secretory response. Calcium response was also repressed in Ag-activated cells defective in Grb2, a major target of phosphorylated NTAL. Unexpectedly, in cells stimulated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) ATPase, the amount of cellular NTAL directly correlated with the uptake of extracellular calcium even though no enhanced tyrosine phosphorylation of NTAL was observed. The combined data indicate that NTAL regulates FcepsilonRI-mediated signaling at multiple steps and by different mechanisms. At early stages NTAL interferes with tyrosine phosphorylation of several substrates and formation of signaling assemblies, whereas at later stages it regulates the activity of store-operated calcium channels through a distinct mechanism independent of enhanced NTAL tyrosine phosphorylation.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• antigeny CD98 - lehké řetězce fyziologie   MeSH
• financování organizované MeSH
• fosforylace MeSH
• intracelulární tekutina metabolismus   MeSH
• krysa rodu rattus MeSH
• mastocyty imunologie   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• nádorové buněčné linie MeSH
• receptory IgE fyziologie   MeSH
• sekvence aminokyselin MeSH
• transportní systém aminokyselin y+ fyziologie   MeSH
• tyrosin metabolismus   MeSH
• vápník metabolismus   MeSH
• vápníková signalizace imunologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH