Abstract
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.
Highlights
The aggregation of high-affinity IgE receptor (FcɛRI)–IgE complexes by multivalent antigen in mast cells leads to the release of a variety of mediators that play important roles in innate and adaptive immunity [1]
We found that cells transduced with individual C-terminal Src kinase (CSK)-specific shRNAs, pooled CSK-specific shRNA (CSK-KD), or CSK-OE displayed an abundance of FcɛRI and c-KIT receptors on the cell surface comparable to corresponding control cells transduced with empty vectors pLKO.1 or pCDH (Figures S1G,H in Supplementary Material)
We found that antigen-stimulated CSK-KD/phosphoprotein associated with glycosphingolipidenriched microdomains (PAG)-KO cells exhibited even more pronounced inhibition of tumor necrosis factor (TNF)-α secretion when compared to pLKO.1/WT cells, indicating positive regulatory role of CSK in bone marrow-derived mast cells (BMMC) cytokine production (Figure 6H)
Summary
The aggregation of high-affinity IgE receptor (FcɛRI)–IgE complexes by multivalent antigen in mast cells leads to the release of a variety of mediators that play important roles in innate and adaptive immunity [1]. The first biochemically well-defined step in FcɛRI-mediated cell activation is tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of FcɛRI β and γ subunits by Src family kinase (SFK) LYN, followed by recruitment of protein tyrosine kinase (PTK) SYK to FcεRI γ and its activation. These mice die in early embryonic stages and their tissues possess Src family tyrosine kinases (SFKs) with increased enzymatic activity [14,15,16]. Mice possessing granulocytes with CSK inactivated by conditional mutagenesis developed acute inflammatory responses [18]
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