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 analyze d the intracellular fate of internalized TCR/CD3 complexes following a ctivation of a CTL clone with an anti-clonotypic mAb (anti-TCR mAb), C onfocal microscopy using fluorescent anti-TCR mAb showed that after 15 min the TCR/CD3 complex colocalized with the transferrin receptor wit hin endosomes, whereas at later times (2 h) it migrated in late endocy tic compartments devoid of transferrin receptor, Using a cell fraction ation technique, CD3 components could be detected in early endosomes i n the absence of ligand-induced internalization, but were detected in late endosomes only after 2-h anti-TCR-induced internalization. In lat e endosomes, the internalized TCR/CD3 complex was found to be associat ed with an active protein kinase, distinct from p56(lck) and p59(fyn), which were mainly present in early endosomes, and ZAP-70, which was o nly present in the postnuclear supernatant, Phosphoamino acid analysis following an in vitro kinase assay of CD3 immunoprecipitates from ear ly and late endosome fractions showed that the CD3 zeta- and epsilon-c hains were phosphorylated exclusively on tyrosine, whereas the CD3 gam ma- and delta-chains were phosphorylated on serine and tyrosine, as we re 40-kDa and 60-kDa associated proteins, Furthermore, the serine phos phorylation was increased in late endosomes compared with early endoso mes, These results suggest that the TCR/CD3 may be associated with dif ferent kinase activities during its intracellular pathway following li gand triggering.