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

Abstract 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 p56 lck and TcR/CD3-associated p59 fyn(T) could account for this cascade, TcR/CD3 driven stimulation of p59 fyn(T) activity has not been demonstrated. In this study, we confirm in Brij 96 based buffers that p59 fyn(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-p59 fyn(T) associated ζ (16–21 kD), p72 (72 kD) and p120/130 (120–130 kD) chains. A distinction between increased recruitment and/or activity of p59 fyn(T) was not possible due to the fact that receptor associated p59 fyn(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 p59 fyn(T) labelling at the autophosphorylation site Tyr420, consistent with stimulated fyn catalytic activity, as well as the phosphorylation of polypeptides at 18–20 (TcRζ), 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.