Transmembrane adaptor proteins (TRAPs) are important organizers and regulators of immunoreceptor-mediated signaling. A bioinformatic search revealed several potential novel TRAPs, including a highly conserved protein, proline rich 7 (PRR7), previously described as a component of the PSD-95/N-methyl-d-aspartate receptor protein complex in postsynaptic densities (PSD) of rat neurons. Our data demonstrate that PRR7 is weakly expressed in other tissues but is readily up-regulated in activated human peripheral blood lymphocytes. Transient overexpression of PRR7 in Jurkat T cell line led to gradual apoptotic death dependent on the WW domain binding motif surrounding Tyr-166 in the intracellular part of PRR7. To circumvent the pro-apoptotic effect of PRR7, we generated Jurkat clones with inducible expression of PRR7 (J-iPRR7). In these cells acute induction of PRR7 expression had a dual effect. It resulted in up-regulation of the transcription factor c-Jun and the activation marker CD69 as well as enhanced production of IL-2 after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. On the other hand, expression of PRR7 inhibited general tyrosine phosphorylation and calcium influx after T cell receptor cross-linking by antibodies. Moreover, we found PRR7 constitutively tyrosine-phosphorylated and associated with Src. Collectively, these data indicate that PRR7 is a potential regulator of signaling and apoptosis in activated T cells.
- MeSH
- adaptorové proteiny signální transdukční biosyntéza genetika imunologie MeSH
- aminokyselinové motivy MeSH
- apoptóza účinky léků fyziologie MeSH
- Caco-2 buňky MeSH
- CD antigeny biosyntéza genetika imunologie MeSH
- diferenciační antigeny T-lymfocytů biosyntéza genetika imunologie MeSH
- fosforylace účinky léků fyziologie MeSH
- HEK293 buňky MeSH
- interleukin-2 biosyntéza genetika imunologie MeSH
- ionofory farmakologie MeSH
- ionomycin farmakologie MeSH
- Jurkat buňky MeSH
- karcinogeny farmakologie MeSH
- krysa rodu rattus MeSH
- lektiny typu C biosyntéza genetika imunologie MeSH
- lidé MeSH
- protoonkogenní proteiny c-jun genetika imunologie metabolismus MeSH
- receptory antigenů T-buněk genetika imunologie metabolismus MeSH
- regulace genové exprese fyziologie MeSH
- T-lymfocyty imunologie metabolismus MeSH
- terciární struktura proteinů MeSH
- tetradekanoylforbolacetát farmakologie MeSH
- U937 buňky MeSH
- vápníková signalizace účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Elevated plasma uric acid indicates an increased risk of cardiovascular diseases associated with endothelial dysfunction. However, the role of uric acid in the pathogenesis of endothelial dysfunction is still a matter of debate. It is not clear whether uric acid is a real causative risk factor, an inert marker, or even a protective molecule with respect to its antioxidant properties. We have studied the effect of uric acid on intact endothelial cells as well as cells with homocysteine-induced endothelial dysfunction. DESIGN: Bovine aortic endothelial cells were treated with uric acid (100 - 600 muM) and homocysteine (100 muM) or with uric acid only. After 24 hours, the cells were stimulated with 1 mug/ml of calcium ionophore A23187, and nitric oxide (NO) production was measured electrochemically with the use of a NO-sensitive microelectrode. The expression of endothelial nitric oxide synthase (eNOS) and eNOS phosphorylation at Ser1179 was estimated with the use of Western blotting. Interaction between NO and uric acid was measured with a NO electrode. Superoxide generation was measured with the use of the fluorescence dye MitoSox Red. RESULTS: Homocysteine strongly diminished A23187-induced NO release. 100 muM uric acid slightly restored NO production; higher concentrations were ineffective. Interestingly, a dose-dependent decrease of NO release was observed in the cells treated only with uric acid. Uric acid did not scavenge NO and did not change eNOS protein expression or phosphorylation at Ser1179, but dose-dependently increased superoxide production in A23187-stimulated cells. CONCLUSION: In conclusion, uric acid decreased NO bioavailability and enhanced superoxide generation in A23187-stimulated bovine aortic endothelial cells.
- MeSH
- aorta MeSH
- calcimycin farmakologie MeSH
- časové faktory MeSH
- endoteliální buňky účinky léků fyziologie MeSH
- fosforylace MeSH
- homocystein metabolismus farmakologie MeSH
- ionofory farmakologie MeSH
- kinetika MeSH
- kultivované buňky MeSH
- kyselina močová metabolismus farmakologie MeSH
- mikroelektrody MeSH
- oxid dusnatý metabolismus MeSH
- skot MeSH
- superoxidy metabolismus MeSH
- synthasa oxidu dusnatého, typ III metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca(2+)-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcepsilonRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcepsilonRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation.
- MeSH
- biologické modely MeSH
- buněčná membrána metabolismus MeSH
- calcimycin farmakologie MeSH
- detergenty farmakologie MeSH
- exocytóza MeSH
- financování organizované MeSH
- fosfatidylseriny chemie MeSH
- fosforylace MeSH
- inhibitory enzymů farmakologie MeSH
- ionofory farmakologie MeSH
- konfokální mikroskopie MeSH
- krysa rodu rattus MeSH
- nádorové buněčné linie MeSH
- proteiny přenášející fosfolipidy metabolismus MeSH
- tyrosin chemie MeSH
- vanadáty farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
The effect of protein kinase C (PKC) inhibitors on porcine oocyte activation by calcium ionophore A23187 was studied. Calcium ionophore applied in a 50 microM concentration for 10 min induced activation in 74% of oocytes matured in vitro. When the ionophore-treated oocytes were exposed to the effect of bisindolylmaleimide I, which inhibits calcium-dependent PKC isotypes (PKC-alpha, -beta(I), -beta(II), -gamma,) and calcium-independent PKC isotypes (PKC-delta, -epsilon), the portion of activated oocytes decreased (at a concentration of 100 nM, 2% of the oocytes were activated). Go6976, the inhibitor of calcium-dependent PKC isotypes PKC-alpha, -beta(I) did not prevent the action of the oocytes treated with calcium ionophore in concentrations from 1 to 100 microM. The inhibitor of PKC-beta(I) and beta(II) isotypes, hispidin, in a concentration of 2 microM-2 mM, was not effective either. The inhibitor of PKC-delta isotype, rottlerin, suppressed activation of the oocytes by calcium ionophore (no oocyte was activated at 10 microM concentration). The PKC-delta isotype in matured porcine oocytes, studied by Western blot analysis, appeared as non-truncated PKC-delta of 77.5 kDa molecular weight, on the one hand, and as truncated PKC-delta, which was present in the form of a doublet of approximately 62.5 and 68 kDa molecular weight, on the other hand. On the basis of these results, it can be supposed that PKC participates in the regulation of processes associated with oocyte activation. Calcium-dependent PKC-alpha, -beta isotypes do not seem to play any significant role in calcium activation. The activation seems to depend on the activity of the calcium-independent PKC-delta isoform.
- MeSH
- acetofenony farmakologie MeSH
- benzopyrany farmakologie MeSH
- calcimycin farmakologie MeSH
- indoly farmakologie MeSH
- inhibitory proteinkinas farmakologie MeSH
- ionofory farmakologie MeSH
- izoenzymy antagonisté a inhibitory fyziologie klasifikace MeSH
- karbazoly farmakologie MeSH
- maleimidy farmakologie MeSH
- oocyty fyziologie účinky léků MeSH
- prasata fyziologie MeSH
- proteinkinasa C antagonisté a inhibitory fyziologie klasifikace MeSH
- pyrony farmakologie MeSH
- vápník fyziologie MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- kazuistiky MeSH
- MeSH
- calcimycin farmakologie MeSH
- finanční podpora výzkumu jako téma MeSH
- hladké svalstvo metabolismus účinky léků MeSH
- hypoxie metabolismus MeSH
- ionofory farmakologie MeSH
- králíci MeSH
- ucho MeSH
- vazodilatancia MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- MeSH
- imunoglobulin G biosyntéza genetika MeSH
- interleukiny sekrece MeSH
- ionofory farmakologie MeSH
- lipopolysacharidy farmakologie MeSH
- myši MeSH
- prezentace antigenu genetika MeSH
- T-lymfocyty pomocné-indukující imunologie MeSH
- tvorba protilátek MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
