Tyrosine and serine protein kinase activities associated with ligand-induced internalized TCR/CD3 complexes.
Ligand engagement of the TCR/CD3 complex leads to its internalization and modulation from the cell surface. In the present study, we analyzed the intracellular fate of internalized TCR/CD3 complexes following activation of a CTL clone with an anti-clonotypic mAb (anti-TCR mAb). Confocal microscopy using fluorescent anti-TCR mAb showed that after 15 min the TCR/CD3 complex colocalized with the transferrin receptor within endosomes, whereas at later times (2 h) it migrated in late endocytic compartments devoid of transferrin receptor. Using a cell fractionation technique, CD3 components could be detected in early endosomes in the absence of ligand-induced internalization, but were detected in late endosomes only after 2-h anti-TCR-induced internalization. In late endosomes, the internalized TCR/CD3 complex was found to be associated with an active protein kinase, distinct from p56(lck) and p59(fyn), which were mainly present in early endosomes, and ZAP-70, which was only present in the postnuclear supernatant. Phosphoamino acid analysis following an in vitro kinase assay of CD3 immunoprecipitates from early and late endosome fractions showed that the CD3 zeta- and epsilon-chains were phosphorylated exclusively on tyrosine, whereas the CD3 gamma- and delta-chains were phosphorylated on serine and tyrosine, as were 40-kDa and 60-kDa associated proteins. Furthermore, the serine phosphorylation was increased in late endosomes compared with early endosomes. These results suggest that the TCR/CD3 may be associated with different kinase activities during its intracellular pathway following ligand triggering.
Luton
F
F
Center of Immunology INSERM-CNRS, Marseille-Luminy, France.
Legendre
V
V
Gorvel
J P
JP
Schmitt-Verhulst
A M
AM
Boyer
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002460:Cell Line; D011992:Endosomes / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase; D019061:src-Family Kinases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D008024:Ligands; D011518:Proto-Oncogene Proteins; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011505:Protein-Tyrosine Kinases; C494960:Fyn protein, mouse; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn; D051746:ZAP-70 Protein-Tyrosine Kinase; C497262:Zap70 protein, mouse; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases