Differential use of BTK and PLC in FcεRI- and KIT-mediated mast cell activation: A marginal role of BTK upon KIT activation.

In mast cells (MCs), the TEC family kinase (TFK) BTK constitutes a central regulator of antigen (Ag)-triggered, FcεRI-mediated PLCγ phosphorylation, Ca²⁺ mobilization, degranulation, and pro-inflammatory cytokine production. Less is known about the function of BTK in the context of stem cell factor (SCF)-induced KIT signaling. In bone marrow-derived MCs (BMMCs), Ag stimulation caused intense phosphorylation of BTK at Y551 in its active center and at Y223 in its SH3-domain, whereas in response to SCF only Y223 was significantly phosphorylated. Further data using the TFK inhibitor Ibrutinib indicated that BTK Y223 is phosphorylated by a non-BTK TFK upon SCF stimulation. In line, SCF-induced PLCγ1 phosphorylation was stronger attenuated by Ibrutinib than by BTK deficiency. Subsequent pharmacological analysis of PLCγ function revealed a total block of SCF-induced Ca²⁺ mobilization by PLC inhibition, whereas only the sustained phase of Ca²⁺ flux was curtailed in Ag-stimulated BMMCs. Despite this severe stimulus-dependent difference in inducing Ca²⁺ mobilization, PLCγ inhibition suppressed Ag- and SCF-induced degranulation and pro-inflammatory cytokine production to comparable extents, suggesting involvement of additional TFK(s) or PLCγ-dependent signaling components. In addition to PLCγ, the MAPKs p38 and JNK were activated by Ag in a BTK-dependent manner; this was not observed upon SCF stimulation. Hence, FcεRI and KIT employ different mechanisms for activating PLCγ, p38, and JNK, which might strengthen their cooperation regarding pro-inflammatory MC effector functions. Importantly, our data clearly demonstrate that analyzing BTK Y223 phosphorylation is not sufficient to prove BTK activation. Copyright © 2019 Elsevier B.V. All rights reserved.

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