Tyrosine phosphorylation of the gamma subunit of Fc gamma receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages.
Fc receptor-mediated phagocytosis in mouse macrophages occurs by a tyrosine kinase-dependent pathway (Greenberg, S., Chang, P., and Silverstein, S.C. (1993) J. Exp. Med. 177, 529-534). To identify proteins that are phosphorylated on tyrosine residues during phagocytosis, we used anti-phosphotyrosine antibodies to perform immunoblotting and immunoprecipitation of lysates derived from Fc receptor-stimulated macrophages. Proteins of 26, 30, 35, 37, 40, 43, 47, 56, 60, 68, 83, 116, and 150 kDa displayed enhanced tyrosine phosphorylation during Fc receptor-mediated phagocytosis. Tyrosine phosphorylation of these proteins was not a consequence of actin polymerization since treatment with cytochalasin D did not alter the pattern of Fc receptor-stimulated protein tyrosine phosphorylation. The 68-kDa tyrosine phosphoprotein was identified as paxillin, a cytoskeletal-associated tyrosine kinase substrate previously identified in fibroblasts and shown to localize to focal adhesions (Turner, C.E., Glenney, J.R., and Burridge, K. (1990) J. Cell Biol. 111, 1059-1068). Paxillin colocalized with F-actin beneath nascent phagosomes. In addition to the above proteins detected by anti-phosphotyrosine immunoblotting, the gamma subunit of FcRI and III was shown to undergo tyrosine phosphorylation during Fc receptor-mediated phagocytosis. Of several candidate tyrosine kinases that may be activated during Fc receptor stimulation, p72syk, but not p125FAK, displayed enhanced tyrosine phosphorylation during Fc receptor aggregation. The coordinated tyrosine phosphorylation of the gamma subunit of macrophage Fc receptors, the tyrosine kinase syk, and the cytoskeletal-associated protein, paxillin, may be important steps in integrating signals between Fc receptors and the underlying cytoskeleton.
Greenberg
S
S
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Chang
P
P
Silverstein
S C
SC
D000199:Actins / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D015638:Cytochalasin D / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D046911:Macromolecular Substances; D017737:Macrophages, Peritoneal / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D008970:Molecular Weight; D051419:Paxillin; D010587:Phagocytosis; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017452:Receptors, IgG / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000199:Actins; D003598:Cytoskeletal Proteins; D046911:Macromolecular Substances; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D017452:Receptors, IgG; D019000:Phosphotyrosine; D015638:Cytochalasin D; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases