Dual functions of Bruton's tyrosine kinase and tec kinase during Fcγ receptor-induced signaling and phagocytosis

Tee family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)gamma, and regulate cytoskeletal rearrangement, yet their role in Fc gamma R-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton's tyrosine kinase (Btk) and Tee, the only Tee kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tee kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited Fc gamma R-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. Fc gamma R-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tee in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLC gamma 2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLC gamma 2 was dependent on Btk and Tee. We found accumulation of diacylglycerol (DAG), a PLC gamma product, in phagosome membranes, and activated Btk, but not Tee, colocalized with phagosomal DAG. Inhibition of Tee family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tee, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tee family kinases participate in at least two stages of Fc gamma R-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tee potentially have distinct roles.