Characterization of the T cell antigen receptor--p60fyn protein tyrosine kinase association by chemical cross-linking.
Engagement of the TCR by specific antigen results in activation of a tyrosine kinase pathway. A candidate for the kinase responsible for the rapid tyrosine phosphorylation detected with T cell activation is p60fyn, a member of the src kinase family. In an earlier study [Samelson et al. (1990) Proc. Natl Acad. Sci. USA 87:4358] this enzyme was co-immunoprecipitated with the TCR from T cells solubilized in digitonin. In that study a sensitive in vitro kinase assay was used to detect the associated p60fyn. It was subsequently found that the reproducibility of the interaction depended on lot-to-lot variations in digitonin. To eliminate the possibility that the association of antigen receptor and kinase is an artifact of solubilization with ill-defined digitonin preparations, a cross-linking protocol was developed to stabilize the interaction between the TCR and p60fyn. T cells were permeabilized with tetanolysin and proteins were cross-linked with the water soluble chemical cross-linker, 3,3' dithiobis(sulfosuccinimidylpropionate). These experiments allowed the confirmation of the interaction between the TCR, p60fyn, and several additional proteins. The cross-linking studies also enabled the mapping of the interaction of p60fyn and associated proteins to the TCR zeta-chain. This technique should have a general use in stabilizing interactions between other receptors and molecules required for intracellular signaling.
Sarosi
G A
GA
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, MD 20982.
Thomas
P M
PM
Egerton
M
M
Phillips
A F
AF
Kim
K W
KW
Bonvini
E
E
Samelson
L E
LE
D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D015151:Immunoblotting; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011948:Receptors, Antigen, T-Cell / Q000302:isolation & purification / Q000378:metabolism*; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*
D003432:Cross-Linking Reagents; D011518:Proto-Oncogene Proteins; D011948:Receptors, Antigen, T-Cell; D011505:Protein-Tyrosine Kinases; C494960:Fyn protein, mouse; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn; D004072:Digitonin