Abstract
We evaluated in Jurkat T cells the time-dependent responses of Fyn, Lck, Syk, and Zap following antibody-mediated cross-linking of the T cell antigen receptor. Our results show that the protein kinase activities of Fyn and Lck were activated within seconds of receptor cross-linking. Fyn activity, as measured by autophosphorylation and tyrosine phosphorylation of an exogenous substrate, was maximal 5 s to 1 min following receptor cross-linking. Lck was also found to be activated within 5 s of antigen receptor cross-linking but differed from Fyn in that Lck activity was elevated for at least 30 min. Syk and Zap protein kinase activities were found to peak between 5 and 10 min following receptor cross-linking, returning to approximately basal activity levels by 60 min. The protein kinase activities of both Syk and Zap were found to parallel their reactivity in immunoblotting experiments with anti-phosphotyrosine antibodies. Both Syk and Zap were found to associate with the tyrosine-phosphorylated zeta subunit of the T cell antigen receptor. These observations imply that T cell antigen receptor signal transduction involves the activation of multiple members of at least two different families of non-transmembrane protein tyrosine kinases.
Highlights
We evaluated in JurkaTt cells the time-dependent re-stimulation, while expressionof kinase-deficientFyn in thymosponses of Fyn,Lck, Syk, and Zap following antibody- cytes results in suppressed T cell antigen receptor (TcR) responsiveness [8]
Our results show that the protein kinase activities of specific T cell line resulted in hyperresponsive TcR-dependent
Syk and Zap protein kiancatsievitieswere found to independent functions as well [12].While not normally thought peak between 5 and 10 min following receptor cross- to interact directly with the TcR,Lck has been clearly doculinking, returning to approximately basal activity levmelesnted to be functionally important for TcR signaling
Summary
Cell Growth and Actiuation-The human leukemic Tcell line Jurkat was usedfor all experiments. The lane rylation in response to anti-TcR antibodyaddition.The cells were labeled Staph representscell lysates with the additioonf formalin-fixed lysed as a function of the indicated times following OKT3 addition and Staphylococcus aureus used for adsorption of immune complexes. Technologies, Inc.) are shown on the righ.t. A and B ) or Lck (panels C and D 1 were immunoprecipitated and used for either immune complex proteinkinaseassays with rabbit muscle enolase as an exogenous substrate (panels A and Co)r used for immunoblotting with anti-Fyn A and E ) ,immune complex kinase assays with GST-Ign as an exogenous substrate (panels R and F ) , immunoblotanalysis probing withAPT antibody (panels C and iGmIm, ourannopabrwlolyoibtsthiinsg eitheranti-Zap (panel D ) or anti-Syk (panel H )antibodies.
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