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.
da Silva
A J
AJ
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA 02115.
Yamamoto
M
M
Zalvan
C H
CH
Rudd
C E
CE
D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008565:Membrane Proteins / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000494:pharmacology*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013997:Time Factors
D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn