Dual functions of Bruton's tyrosine kinase and Tec kinase during Fcgamma receptor-induced signaling and phagocytosis.
Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)gamma, and regulate cytoskeletal rearrangement, yet their role in FcgammaR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton's tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec 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 FcgammaR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcgammaR-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 Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLCgamma2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCgamma2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCgamma product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcgammaR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.
Toronto General Research Institute, University Health Network, Department of Immunology, University of Toronto, Toronto, Ontario, Canada. jbilen@uhnres.utoronto.ca
Jenny
J
Jongstra-Bilen
Adrianet
A
Puig Cano
Manvi
M
Hasija
Haiyan
H
Xiao
C I Edvard
CI
Smith
Myron I
MI
Cybulsky
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D002460:Cell Line; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D016177:Macrophage-1 Antigen / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010587:Phagocytosis / Q000276:immunology*; D010588:Phagosomes / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D017452:Receptors, IgG / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology*
D004075:Diglycerides; D016177:Macrophage-1 Antigen; D034741:RNA, Small Interfering; D017452:Receptors, IgG; C089848:Tec protein-tyrosine kinase; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625951:Btk protein, mouse