Signaling through CD16b in human neutrophils involves the Tec family of tyrosine kinases

Tee kinases belong to the second largest family of nonreceptor tyrosine kinases. Although these kinases are expressed in myeloid cells, little is known about their implication in neutrophil function. We recently reported the participation of Tee kinases in the responses of human neutrophils to the bacterial peptide N-formyl-1-methionyl-1-leucyl-1-phenylalanine via G-coupled protein receptors. In this study, we extended our investigations of Tee kinases to the signaling of the glycosylphosphatidylinositol-linked receptor CD16b, which is highly and specifically expressed in neutrophils. The results obtained indicate that Tee is translocated to the plasma membrane, phosphorylated, and activated upon CD16b cross-linking and that the activation of Tee is inhibited by Src-specific inhibitors as well as by the phosphatidylinositol-3 kinase inhibitor, wortmannin. As no specific inhibitor of Tee exists, the role of Tee kinases was further investigated using a-Cyano-b-hydroxy-b-methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), a compound known to inhibit Bruton's tyrosine kinase. We show that this compound also inhibits the kinase activity of Tee and provide evidence that the mobilization of intracellular calcium and the tyrosine phosphorylation of phospholipase C gamma 2 (PLC gamma 2) induced upon CD16b engagement are inhibited by LFM-A13. We also show that Tee kinases are important for CD16b-dependent degranulation of neutrophils. In summary, we provide direct evidence for the implication of Tee in CD16b signaling and suggest that Tee kinases are involved in the phosphorylation and activation of PLC gamma 2 and subsequently, in the mobilization of calcium in human neutrophils.