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.

• Klíčová slova
• C-terminal Src kinase, LYN, SHP-1, STAT5, cytokines, degranulation, mast cell, phosphoprotein associated with glycosphingolipid-enriched microdomains,
• 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
• Názvy látek
• C-terminální Src kinasa MeSH
• CSK protein, human MeSH Prohlížeč
• cytokiny MeSH
• fibronektiny MeSH
• fosfoproteiny MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• membránové proteiny MeSH
• mezibuněčné signální peptidy a proteiny MeSH
• Pag protein, mouse MeSH Prohlížeč
• Pag1 protein, mouse MeSH Prohlížeč
• Ptpn6 protein, mouse MeSH Prohlížeč
• receptory IgE MeSH
• skupina kinas odvozených od src-genu MeSH
• transkripční faktor STAT5 MeSH
• tyrosin MeSH
• tyrosinfosfatasa nereceptorového typu 6 MeSH
• vápník MeSH

The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved.

• MeSH
• anafylaxe genetika   MeSH
• buňky kostní dřeně metabolismus   fyziologie   MeSH
• C-terminální Src kinasa MeSH
• degranulace buněk MeSH
• fosfolipasy typu C metabolismus   MeSH
• fosfoproteiny genetika   MeSH
• fosforylace MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• kinasa Syk MeSH
• malá interferující RNA MeSH
• mastocyty metabolismus   fyziologie   MeSH
• membránové proteiny genetika   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• protoonkogenní proteiny c-fyn biosyntéza   MeSH
• protoonkogenní proteiny c-kit metabolismus   MeSH
• receptory IgE metabolismus   MeSH
• RNA interference MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu biosyntéza   metabolismus   MeSH
• tyrosinkinasy metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• C-terminální Src kinasa MeSH
• fosfolipasy typu C MeSH
• fosfoproteiny MeSH
• Fyn protein, mouse MeSH Prohlížeč
• intracelulární signální peptidy a proteiny MeSH
• kinasa Syk MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• malá interferující RNA MeSH
• membránové proteiny MeSH
• Pag1 protein, mouse MeSH Prohlížeč
• protoonkogenní proteiny c-fyn MeSH
• protoonkogenní proteiny c-kit MeSH
• receptory IgE MeSH
• skupina kinas odvozených od src-genu MeSH
• Syk protein, mouse MeSH Prohlížeč
• tyrosinkinasy MeSH
• vápník MeSH