Engagement of the TcR/CD3 complex stimulates p59fyn(T) activity: detection of associated proteins at 72 and 120-130 kD.

Engagement of the T cell antigen-receptor complex (TcR/CD3) induces the rapid tyrosine phosphorylation of a spectrum of substrates whose modification is crucial to the activation process. Although CD4-associated p56lck and TcR/CD3-associated p59fyn(T) could account for this cascade, TcR/CD3 driven stimulation of p59fyn(T) activity has not been demonstrated. In this study, we confirm in Brij 96 based buffers that p59fyn(T) can be co-purified in association with the TcR/CD3 complex, and further demonstrate that antibody-induced cross-linking of TcR/CD3 on the cell surface results in a dramatic increase in the detection of receptor associated kinase activity. This results in an increased phosphorylation and detection of TcR/CD3-p59fyn(T) associated zeta (16-21 kD), p72 (72 kD) and p120/130 (120-130 kD) chains. A distinction between increased recruitment and/or activity of p59fyn(T) was not possible due to the fact that receptor associated p59fyn(T) could not be detected by immunoblotting. However, an alternative approach using membrane vesicles demonstrated an anti-CD3 mediated induced increase (2-5-fold) in the phosphorylation of the fyn kinase. Augmented catalytic activity was accompanied by p59fyn(T) labelling at the autophosphorylation site Tyr420, consistent with stimulated fyn catalytic activity, as well as the phosphorylation of polypeptides at 18-20 (TcR zeta), 31, 90 and 130 kD. Stimulation of fyn activity implicates this kinase as a mediator of the tyrosine phosphorylation events originating from the TcR/CD3 complex.

Biochemical identification of a direct physical interaction between the CD4:p56lck and Ti(TcR)/CD3 complexes.

The CD4 and CD8 antigens function in synergy with the TcR/CD3 complex in the generation of intracellular signals leading to T cell proliferation. The association of the protein-tyrosine kinase p56lck with CD4 and CD8 provides a potential mechanism in the generation of intracellular signals. Several studies have shown that CD4 can co-modulate with TcR/CD3 suggesting that these receptor complexes may associated on the surface of the T cell. Nevertheless, it has proven difficult to formally demonstrate a direct physical interaction between the CD4 and TcR/CD3 complexes using biochemical techniques. In this study, we have used the sensitivity of the in vitro kinase assay to show a direct physical linkage between the CD4:p56lck complex and various CD3 subunits. Immunoprecipitation of CD4 from cell lysates derived from the T lymphoblastoid line HPB-ALL results in the co-purification of p56lck with an additional polypeptide at 20 kDa. Re-precipitation analysis and isoelectric focusing demonstrated that this band corresponds to the CD3 epsilon chain. An alternative approach which involves the labeling of microsomal membranes with [gamma-32P]ATP revealed the presence of CD3 epsilon and zeta chains in anti-CD4 immunoprecipitates. By contrast, we were unable to demonstrate the association of the CD4:p56lck and TcR/CD3 complex in resting peripheral blood lymphocytes. These data indicate that the CD4:p56lck and TcR/CD3 complexes have the ability to form stable complexes on the surface of certain T cell lines.