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.

Department of Signal Transduction Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

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