Abstract
The protein tyrosine kinase Syk is activated upon engagement of immune recognition receptors. We have focused on the identification of signaling elements immediately downstream to Syk in the pathway leading to T cell activation. To circumvent T cell receptor (TCR). CD3 activation of Src family kinases, we constructed a signaling molecule with an extracellular single chain Fv of an anti-TNP antibody, attached via a transmembrane region to Syk (scFv-Syk). In a murine T cell hybridoma, direct aggregation of chimeric Syk with antigen culminates in interleukin-2 production and target cell lysis. Initially, it causes an increase in the association between scFv-Syk and the cytosolic protein Cbl and subsequently promotes tyrosine phosphorylation of Cbl. Interestingly, although both Cbl and phospholipase C-gamma (PLC-gamma) are phosphorylated in this hybridoma upon TCR.CD3 cross-linking, these two events are uncoupled in scFv-Syk-transfected cells, in which we were unable to detect antigen-driven PLC-gamma phosphorylation. These results support a model in which Syk can initiate and directly activate the T cell's signaling machinery and position Cbl as a primary tyrosine kinase substrate in this pathway. Furthermore, for efficient PLC-gamma phosphorylation to occur in these cells, the combined actions of different tyrosine kinase families may be required.
Highlights
Hematopoietic lineages, such as B cells, myeloid cells, and thymocytes [6]
DNA was transfected into a mutant of the murine T cell hybridoma MD45, named 27J, which lacks the T cell receptor as a result of defective production of its ␣ chain [29]
Given the importance of Syk kinase activity in lymphocyte activation [2, 16], identification of its early targets of phosphorylation and association in T cells is an immediate goal for understanding propagation of the signal leading to T cell activation
Summary
Upon separation by SDS-PAGE under reducing conditions and transfer of proteins to nitrocellulose, immunoblotting was performed with the monoclonal anti-phosphotyrosine antibody 4G10 followed by detection with peroxidase-labeled goat anti-mouse antibody and ECL. Anti-Sp6 antibody GK20.5, first bound to protein G-Sepharose, was used for immunoprecipitation of chimeric Syk through the scFv portion. Kinase assays were performed on anti-Sp6 immunoprecipitates, which were washed three times in a buffer containing 50 mM Hepes, pH 7, 150 mM NaCl, 0.1% Triton, 10% glycerol, 1 mM Na3VO4, and 5 mM NaF. To measure specific IL-2 production, 105 transfectants expressing the scFv-Syk chimera were incubated with 3 ϫ 105 TNP-modified A.20 or L1210 B lymphoma cells in DMEM containing 10% fetal calf serum for 18 –24 h. To evaluate the ability of transfectants to lyse TNP-labeled target cells, the 51Cr-release assay was performed over 4 – 8 h [26]
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