T cell receptor/CD3 complex internalization following activation of a cytolytic T cell clone: Evidence for a protein kinase C‐independent staurosporine‐sensitive step

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Abstract
The fate of the T cell receptor (TcR)/CD3 complex was examined on a cytotoxic T lymphocyte (CTL) clone (KB5.C20) activated either via binding of an anti-TcR monoclonal antibody (mAb) or by a Ca2+ ionophore and phorbol 12-myristate 13-acetate (PMA). After binding of the anti-TcR mAb, electron microscopy revealed internalization through coated vesicles followed by slow degradation of the antibody as shown by use of radiolabeled mAb. The influence of activation on TcR/CD3 internalization was analyzed. The Ca2+ ionophore alone had no effect on internalization, whereas PMA induced an accelerated internalization of anti-TcR mAb. PMA-induced internalization was dependent on protein kinase C (PKC) as shown by its absence in PKC-depleted cells or in the presence of the PKC inhibitor staurosporine. Anti-TcR mAb-induced internalization was maintained in PKC-depleted cells, but unexpectedly remained sensitive to inhibition by staurosporine. The monovalent anti-TcR mAb Fab fragment is non-stimulatory for the CTL. It was poorly internalized but its internalization was induced by PMA. Surprisingly, on PKC-depleted cells, the Fab was internalized more readily than in untreated cells and this internalization was sensitive to inhibition by staurosporine. Inhibition of PMA-induced phosphorylation of γ and ζ subunits of CD3 was demonstrated after depletion of PKC or in the presence of staurosporine, confirming that PKC function was inhibited in those conditions. Cross-linking of the TcR via plastic-coated anti-TcR mAb led to phosphorylation of CD3 γ and ϵ and also of ζ, known to be phosphorylated on tyrosines. All of these phosphorylation events were inhibited by treatment with staurosporine. Our results indicate that staurosporine inhibits the receptor internalization induced by anti-TcR mAb by means other than inhibition of PKC, suggesting that other kinases may control a step of this internalization process.