C-terminal Src kinase (CSK) is a major negative regulator of Src family tyrosine kinases (SFKs) that play critical roles in immunoreceptor signaling. CSK is brought in contiguity to the plasma membrane-bound SFKs via binding to transmembrane adaptor PAG, also known as CSK-binding protein. The recent finding that PAG can function as a positive regulator of the high-affinity IgE receptor (FcεRI)-mediated mast cell signaling suggested that PAG and CSK have some non-overlapping regulatory functions in mast cell activation. To determine the regulatory roles of CSK in FcεRI signaling, we derived bone marrow-derived mast cells (BMMCs) with reduced or enhanced expression of CSK from wild-type (WT) or PAG knockout (KO) mice and analyzed their FcεRI-mediated activation events. We found that in contrast to PAG-KO cells, antigen-activated BMMCs with CSK knockdown (KD) exhibited significantly higher degranulation, calcium response, and tyrosine phosphorylation of FcεRI, SYK, and phospholipase C. Interestingly, FcεRI-mediated events in BMMCs with PAG-KO were restored upon CSK silencing. BMMCs with CSK-KD/PAG-KO resembled BMMCs with CSK-KD alone. Unexpectedly, cells with CSK-KD showed reduced kinase activity of LYN and decreased phosphorylation of transcription factor STAT5. This was accompanied by impaired production of proinflammatory cytokines and chemokines in antigen-activated cells. In line with this, BMMCs with CSK-KD exhibited enhanced phosphorylation of protein phosphatase SHP-1, which provides a negative feedback loop for regulating phosphorylation of STAT5 and LYN kinase activity. Furthermore, we found that in WT BMMCs SHP-1 forms complexes containing LYN, CSK, and STAT5. Altogether, our data demonstrate that in FcεRI-activated mast cells CSK is a negative regulator of degranulation and chemotaxis, but a positive regulator of adhesion to fibronectin and production of proinflammatory cytokines. Some of these pathways are not dependent on the presence of PAG.
- MeSH
- analýza rozptylu MeSH
- buňky kostní dřeně fyziologie MeSH
- C-terminální Src kinasa MeSH
- cytokiny metabolismus MeSH
- degranulace buněk MeSH
- fibronektiny metabolismus MeSH
- fosfoproteiny metabolismus MeSH
- fosforylace MeSH
- genetické vektory MeSH
- HEK293 buňky MeSH
- lidé MeSH
- mastocyty fyziologie MeSH
- membránové proteiny metabolismus MeSH
- mezibuněčné signální peptidy a proteiny MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- receptory IgE metabolismus MeSH
- signální transdukce imunologie MeSH
- skupina kinas odvozených od src-genu metabolismus fyziologie MeSH
- transkripční faktor STAT5 metabolismus MeSH
- tyrosin metabolismus MeSH
- tyrosinfosfatasa nereceptorového typu 6 metabolismus MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in virulence of Bordetella pertussis. CyaA penetrates myeloid cells expressing the complement receptor 3 (αM β2 integrin CD11b/CD18) and subverts bactericidal capacities of neutrophils and macrophages by catalysing unregulated conversion of cytosolic ATP to the key signalling molecule adenosine 3',5'-cyclic monophosphate (cAMP). We show that the signalling of CyaA-produced cAMP hijacks, by an as yet unknown mechanism, the activity of the tyrosine phosphatase SHP-1 and activates the pro-apoptotic BimEL-Bax cascade. Mitochondrial hyperpolarization occurred in human THP-1 macrophages within 10 min of exposure to low CyaA concentrations (e.g. 20 ng ml(-1) ) and was accompanied by accumulation of BimEL and association of the pro-apoptotic factor Bax with mitochondria. BimEL accumulation required cAMP/protein kinase A signalling, depended on SHP-1 activity and was selectively inhibited upon small interfering RNA knockdown of SHP-1 but not of the SHP-2 phosphatase. Moreover, signalling of CyaA-produced cAMP inhibited the AKT/protein kinase B pro-survival cascade, enhancing activity of the FoxO3a transcription factor and inducing Bim transcription. Synergy of FoxO3a activation with SHP-1 hijacking thus enables the toxin to rapidly trigger a persistent accumulation of BimEL, thereby activating the pro-apoptotic programme of macrophages and subverting the innate immunity of the host.
- MeSH
- adenylátcyklasový toxin metabolismus MeSH
- AMP cyklický metabolismus MeSH
- apoptóza fyziologie MeSH
- Bordetella pertussis metabolismus patogenita MeSH
- fagocyty metabolismus mikrobiologie MeSH
- forkhead transkripční faktory genetika metabolismus MeSH
- interakce hostitele a patogenu fyziologie MeSH
- lidé MeSH
- makrofágy metabolismus MeSH
- membránové proteiny metabolismus MeSH
- mitochondrie metabolismus MeSH
- protein X asociovaný s bcl-2 metabolismus MeSH
- proteiny regulující apoptózu metabolismus MeSH
- protoonkogenní proteiny metabolismus MeSH
- signální transdukce MeSH
- tyrosinfosfatasa nereceptorového typu 6 metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates complement receptor 3-expressing phagocytes and catalyzes uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by complement-dependent oxidative burst and opsonophagocytic mechanisms. We show that cAMP signaling through the protein kinase A (PKA) pathway activates Src homology domain 2 containing protein tyrosine phosphatase (SHP) 1 and suppresses production of bactericidal NO in macrophage cells. Selective activation of PKA by the cell-permeable analog N(6)-benzoyladenosine-3',5'-cyclic monophosphate interfered with LPS-induced inducible NO synthase (iNOS) expression in RAW264.7 macrophages, whereas inhibition of PKA by H-89 largely restored the production of iNOS in CyaA-treated murine macrophages. CyaA/cAMP signaling induced SHP phosphatase-dependent dephosphorylation of the c-Fos subunit of the transcription factor AP-1 and thereby inhibited TLR4-triggered induction of iNOS gene expression. Selective small interfering RNA knockdown of SHP-1, but not of the SHP-2 phosphatase, rescued production of TLR-inducible NO in toxin-treated cells. Finally, inhibition of SHP phosphatase activity by NSC87877 abrogated B. pertussis survival inside murine macrophages. These results reveal that an as yet unknown cAMP-activated signaling pathway controls SHP-1 phosphatase activity and may regulate numerous receptor signaling pathways in leukocytes. Hijacking of SHP-1 by CyaA action then enables B. pertussis to evade NO-mediated killing in sentinel cells of innate immunity.
- MeSH
- adenylátcyklasový toxin imunologie MeSH
- aktivace enzymů imunologie MeSH
- AMP cyklický MeSH
- Bordetella pertussis imunologie MeSH
- buněčné linie MeSH
- infekce bakteriemi rodu Bordetella enzymologie imunologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- makrofágy imunologie mikrobiologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- oxid dusnatý biosyntéza MeSH
- signální transdukce imunologie MeSH
- tyrosinfosfatasa nereceptorového typu 6 imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transmembrane adaptor proteins are membrane-anchored proteins consisting of a short extracellular part, a transmembrane domain, and a cytoplasmic part with various protein-protein interaction motifs but lacking any enzymatic activity. They participate in the regulation of various signaling pathways by recruiting other proteins to the proximity of cellular membranes where the signaling is often initiated and propagated. In this work, we show that LST1/A, an incompletely characterized protein encoded by MHCIII locus, is a palmitoylated transmembrane adaptor protein. It is expressed specifically in leukocytes of the myeloid lineage, where it localizes to the tetraspanin-enriched microdomains. In addition, it binds SHP-1 and SHP-2 phosphatases in a phosphotyrosine-dependent manner, facilitating their recruitment to the plasma membrane. These data suggest a role for LST1/A in negative regulation of signal propagation.
- MeSH
- buněčná membrána metabolismus MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- hlavní histokompatibilní komplex fyziologie MeSH
- Jurkat buňky MeSH
- lidé MeSH
- membránové proteiny chemie genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- myeloidní buňky cytologie metabolismus MeSH
- plakiny metabolismus MeSH
- primární buněčná kultura MeSH
- pseudopodia metabolismus MeSH
- sekvence aminokyselin MeSH
- signální transdukce fyziologie MeSH
- terciární struktura proteinů fyziologie MeSH
- transport proteinů fyziologie MeSH
- tyrosinfosfatasa nereceptorového typu 11 metabolismus MeSH
- tyrosinfosfatasa nereceptorového typu 6 metabolismus MeSH
- U937 buňky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hypermethylation of CpG islands within gene promoters is one of various mechanisms of gene silencing involved in the pathogenesis of human cancer. By using methylation-specific polymerase chain reaction we explored aberrant promoter methylation of five tumour suppressor genes in 29 patients with chronic lymphocytic leukaemia. Aberrant methylation of DLC1, SHP1, p15 and p16 occurred, respectively, in 89.7 %, 70 %, 62.1 % and 31 % of patients at diagnosis. Lamin A/C was unmethylated in all the samples. Hypermethylation of at least one gene was detected in 96.6 % of patients. Concurrent methylation of two or more genes correlated with Rai stage at diagnosis.
- MeSH
- chronická lymfatická leukemie diagnóza genetika patologie MeSH
- CpG ostrůvky MeSH
- DNA nádorová genetika metabolismus MeSH
- dospělí MeSH
- financování organizované MeSH
- inhibitor p15 cyklin-dependentní kinasy genetika metabolismus MeSH
- inhibitor p16 cyklin-dependentní kinasy genetika metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- metylace DNA MeSH
- nádorové supresorové proteiny genetika metabolismus MeSH
- polymerázová řetězová reakce metody MeSH
- promotorové oblasti (genetika) MeSH
- proteiny aktivující GTPasu genetika metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- senioři MeSH
- tumor supresorové geny fyziologie MeSH
- tyrosinfosfatasa nereceptorového typu 6 genetika metabolismus MeSH
- umlčování genů MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH