Evidence for protein tyrosine kinase involvement in ligand-induced TCR/CD3 internalization and surface redistribution.
Triggering of the TCR/CD3 complex can lead to its internalization and modulation from the cell surface. In the present study, we address the question of the dependence of internalization on protein tyrosine kinase (PTK) activation. With use of an activating anti-clonotypic (anti-Ti) mAb on a CTL clone, we have shown that the PTK inhibitors genistein and tyrphostin 25 delayed anti-Ti-induced internalization, but did not affect fluid phase protein uptake or transferrin receptor cycling. Confocal microscopy with use of fluorescent anti-Ti mAb revealed that the inhibition of TCR internalization corresponded to the induction of large patches that were localized in cell membrane areas depleted of polymerized actin, the formation of which was dependent on the combined action of the anti-Ti mAb and the PTK inhibitors. In contrast to the effect of these PTK inhibitors, depletion of Src-like PTKs by T cell pretreatment with herbimycin A led to an increased rate of anti-Ti-induced internalization. Internalization induced by the monovalent Fab fraction of anti-Ti mAb was similarly affected by the PTK inhibitors, although the extent of induced internalization was less by approximately one-half. An analysis of substrates phosphorylated in kinase assays on TCR/CD3 immunoprecipitates of the CTL, which were activated by anti-Ti mAb in both the absence and presence of genistein, identified protein bands in which phosphorylation or association with CD3 was inhibited in the presence of genistein. Thus, a genistein-sensitive PTK activity seems to control ligand-induced TCR/CD3 complex redistribution and internalization.
Luton
F
F
Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, France.
Buferne
M
M
Davoust
J
J
Schmitt-Verhulst
A M
AM
Boyer
C
C
D000199:Actins / Q000378:metabolism; D000818:Animals; D017252:CD3 Complex / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000648:ultrastructure; D015536:Down-Regulation; D004705:Endocytosis; D007152:Immunologic Capping; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism
D000199:Actins; D017252:CD3 Complex; D008024:Ligands; D011948:Receptors, Antigen, T-Cell; D011505:Protein-Tyrosine Kinases