Tyrosine phosphorylation of proteins plays a central role in T cell activation. Mitogens or anti-receptor antibodies have been employed to study these signaling events, but the extent to which these mimic receptor interactions with native ligands is unclear, Cytotoxic T lymphocytes can be activated for functional responses using purified, native class I ligands presented on a surface. Previous work showed that stimulation with fluid-phase anti-T cell receptor (TCR) monoclonal antibody (mAb) activates CDS to mediate adhesion to class I proteins and that activated CD8 generates a co-stimulatory signal upon binding to class I. Changes in tyrosine phosphorylation of substrates and activity of the p56(lck) kinase have now been examined in this two-step process, The observed changes are small in comparison to those found using more potent nonphysiological stir-null, but may more accurately reflect the events required for activation of functional responses. Fluid-phase anti-TCR mAb caused increased tyrosine phosphorylation of a discrete subset of cellular substrates. Increased phosphorylation of additional substrates occurred upon CD8 binding to class I, resulting in a phosphorylation pattern comparable to that found in cells stimulated with class I alloantigen. Anti-TCR mAb alone caused increased tyrosine phosphorylation of p56(lck). When CDS bound to class I, phosphorylation of p56(lck) decreased to below the basal level Pound in unstimulated cells, accompanied by a substantial increase in kinase activity, These results are consistent with the two-step model for TCR activation of CD8/class I interactions and directly demonstrate that CD8 binding to class I leads to up-regulation of p56(lck) activity.
