Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination.

• MeSH
• adaptorové proteiny signální transdukční imunologie   metabolismus   MeSH
• aktivace lymfocytů * MeSH
• fosfolipasa C gama metabolismus   MeSH
• fosforylace imunologie   MeSH
• ligandy MeSH
• membránové proteiny imunologie   metabolismus   MeSH
• myši MeSH
• protein-tyrosinkináza ZAP-70 metabolismus   MeSH
• receptory antigenů T-buněk imunologie   metabolismus   MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• tyrosin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

We evaluated the impact of ataxia-telangiectasia mutated kinase inhibitor KU55933, DNA-dependent protein kinase inhibitor NU7441 and ataxia telangiectasia and rad3-related kinase inhibitor VE821 in human peripheral lymphocytes in vitro. The lymphocytes were divided into 5 groups: non-irradiated control, irradiated group (2 Gy) and 3 groups pretreated with inhibitors 30 min before irradiation. We used flow cytometry to evaluate phosphorylated H2AX (γ-H2AX) and cytotoxicity (Apoptest). Micronucleus assay was used to assess genotoxicity. After irradiation, γ-H2AX, incidence of micronuclei (MN), nucleoplasmatic bridges (NPBs) and nuclear buds in binuclear cells, MN in mononuclear cells and apoptosis were increased. KU55933 decreased γ-H2AX and inhibited ionizing radiation-induced cytotoxicity. NU7441 showed no effect on γ-H2AX but it significantly increased MN and NPBs in binuclear cells and apoptosis. VE821 decreased γ-H2AX, whereas genotoxicity and cytotoxicity were not affected. In conclusion, KU55933 protected lymphocytes, which might be employed to preserve the immune system during anticancer therapy. NU7441 radiosensitized lymphocytes, thus, undesirable side effects toward immune system could be expected. VE821 showed decrease of γ-H2AX with no radiosensitizing effects in our model likely due to p53 positive status, which underlies the concept of its application in p53 negative environment.

• Klíčová slova
• Účinky ionizujícího záření způsobené inhibitory ATM (KU55933), DNA-PK (NU7441) a ATR (VE821) na lymfocyty periferní krve,
• MeSH
• ATM protein účinky záření   MeSH
• biomedicínský výzkum MeSH
• financování organizované MeSH
• fosfatidylinositol-3-kinasy antagonisté a inhibitory   účinky záření   MeSH
• fosforylace imunologie   účinky záření   MeSH
• histony chemie   účinky záření   MeSH
• ionizující záření * MeSH
• krevní a imunitní systémy cytologie   imunologie   účinky záření   MeSH
• lidé MeSH
• lymfocyty * cytologie   imunologie   účinky záření   MeSH
• statistika jako téma MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH

OBJECTIVE: The aberrant expression of myeloid antigens on acute lymphoblastic leukemia (ALL) cells is a well-documented phenomenon. So far, there have been no reports of a functional consequence of this aberrant expression. The granulocytic marker carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6, CD66c) is a GPI-anchored molecule that is reported to be the most frequently aberrantly expressed myeloid marker in ALL with a strong correlation with genotype. MATERIALS AND METHODS: We mimicked CEACAM6 signaling in ALL cells by cross-linking with anti-CEACAM6 antibody. Next, we measured a response to CEACAM6 signaling by integrin subunits expression, integrin ligand binding, phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), Akt, and p38 mitogen-activated protein kinase (MAPK) and apoptosis by flow cytometry. RESULTS: Following CEACAM6 cross-linking in ALL cells, we detected Erk1/2, Akt, and p38 MAPK phosphorylation and integrin upregulation, as well as enhanced binding of integrin ligands (vascular cell adhesion molecule-1 [VCAM-1] and intercellular cell adhesion molecule-1 [ICAM-1]). However, CEACAM6 signaling resulted in an increase in apoptosis, unlike other GPI-anchored molecules, such as CD24. CONCLUSION: The present study is the first to demonstrate the functional consequences of CEACAM6 cross-linking in B-cell precursor ALL cells.

• MeSH
• antigeny nádorové imunologie   metabolismus   MeSH
• apoptóza MeSH
• CD antigeny imunologie   metabolismus   MeSH
• fosforylace účinky léků   imunologie   MeSH
• GPI-vázané proteiny MeSH
• imunologický capping MeSH
• integriny imunologie   metabolismus   MeSH
• lidé MeSH
• MAP kinasový signální systém MeSH
• mezibuněčná adhezivní molekula-1 imunologie   metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 3 imunologie   metabolismus   MeSH
• mitogenem aktivované proteinkinasy p38 imunologie   metabolismus   MeSH
• molekuly buněčné adheze antagonisté a inhibitory   imunologie   metabolismus   MeSH
• nádorové buněčné linie MeSH
• onkogenní protein v-akt imunologie   metabolismus   MeSH
• pre-B-buněčná leukemie imunologie   metabolismus   MeSH
• protilátky nádorové imunologie   farmakologie   MeSH
• regulace genové exprese u leukemie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The earliest known biochemical step that occurs after ligand binding to the multichain immune recognition receptor is tyrosine phosphorylation of the receptor subunits. In mast cells and basophils activated by multivalent antigen-IgE complexes, this step is mediated by Src family kinase Lyn, which phosphorylates the high affinity IgE receptor (Fc epsilonRI). However, the exact molecular mechanism of this phosphorylation step is incompletely understood. In this study, we tested the hypothesis that changes in activity and/or topography of protein-tyrosine phosphatases (PTPs) could play a major role in the Fc epsilonRI triggering. We found that exposure of rat basophilic leukemia cells or mouse bone marrow-derived mast cells to PTP inhibitors, H(2)O(2) or pervanadate, induced phosphorylation of the Fc epsilonRI subunits, similarly as Fc epsilonRI triggering. Interestingly, and in sharp contrast to antigen-induced activation, neither H(2)O(2) nor pervanadate induced any changes in the association of Fc epsilonRI with detergent-resistant membranes and in the topography of Fc epsilonRI detectable by electron microscopy on isolated plasma membrane sheets. In cells stimulated with pervanadate, H(2)O(2) or antigen, enhanced oxidation of active site cysteine of several PTPs was detected. Unexpectedly, most of oxidized phosphatases bound to the plasma membrane were associated with the actin cytoskeleton. Several PTPs (SHP-1, SHP-2, hematopoietic PTP, and PTP-MEG2) showed changes in their enzymatic activity and/or oxidation state during activation. Based on these and other data, we propose that down-regulation of enzymatic activity of PTPs and/or changes in their accessibility to the substrates play a key role in initial tyrosine phosphorylation of the Fc epsilonRI and other multichain immune receptors.

• MeSH
• aktivace enzymů účinky léků   genetika   imunologie   MeSH
• antigeny imunologie   metabolismus   farmakologie   MeSH
• fosforylace účinky léků   genetika   imunologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• krysa rodu rattus MeSH
• mastocyty imunologie   metabolismus   MeSH
• membránové mikrodomény genetika   imunologie   metabolismus   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oxidace-redukce účinky léků   MeSH
• oxidancia farmakologie   MeSH
• peroxid vodíku farmakologie   MeSH
• receptory IgE genetika   imunologie   metabolismus   MeSH
• skupina kinas odvozených od src-genu genetika   imunologie   metabolismus   MeSH
• transport proteinů účinky léků   genetika   imunologie   MeSH
• tyrosinfosfatasy antagonisté a inhibitory   genetika   imunologie   metabolismus   MeSH
• vanadáty farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Klíčová slova
• aktivace signální dráhy JAK/STAT,
• MeSH
• fosforylace genetika   imunologie   účinky léků   MeSH
• interferony genetika   imunologie   účinky léků   MeSH
• Janus kinasy genetika   imunologie   účinky léků   MeSH
• lékařská onkologie metody   trendy   MeSH
• lidé MeSH
• nádorové buněčné linie * imunologie   účinky léků   MeSH
• nádory prsu * genetika   imunologie   MeSH
• serin metabolismus   účinky léků   MeSH
• transkripční faktory STAT * genetika   imunologie   účinky léků   MeSH
• tyrosin metabolismus   účinky léků   MeSH
• western blotting metody   využití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH