Abstract
The earliest biochemical event known to be associated with T cell receptor (TCR) engagement is the activation of a protein tyrosine kinase thought to be a member of the src family. Expression of CD45, the major receptor tyrosine phosphatase present on T cells, is required for efficient coupling between the TCR and its signaling machinery. One model to explain the role of CD45 in regulating TCR signaling is that the phosphatase dephosphorylates the regulatory C-terminal tyrosine of lck. In the present report we confirm the finding of a trimolecular complex containing CD45, lck, and a 34-kDa protein (p34) in the Jurkat T cell line. Additionally, we extend this work with the observation that specific in vitro kinase activity associated with CD45 requires the expression of lck in Jurkat-derived clones as does the in vivo phosphorylation of p34. The association between CD45 and lck is shown not to require the expression of or activation of the TCR. Finally, we demonstrate that even in the absence of lck p34 associates with CD45, implying a direct association between this molecule and the phosphatase. These data suggest strongly that lck is the relevant protein tyrosine kinase found in CD45 immunoprecipitates in the Jurkat T cell line and that there is an additional association between CD45 and p34 which does not require the presence of the protein tyrosine kinase.
Highlights
The earliest biochemical event known to be associated with T cell receptor (TCR) engagement is the activation of a protein tyrosine kinasethought to be a memberof the src family
None of the various components of the TCR has intrinsic kinase activity, it is necessary for the TCR to recruit a protein tyrosine kinasefor this function
These data suggest strongly that lck is the relevant protein tyrosine kinase found in CD45 immunoprecipitates in the Jurkat T cell line and that there is an additional association between CD45 and p34 which does not require the presence of the protein tyrosine kinase
Summary
Cell Lines-Each of the cell lines is a derivative of the E6-1 clone of the Jurkat human T cell leukemic line (American Type Culture Collection). MonoclonalAntibodies ( d b s ) and Antisera-Anti-CD45, mAbs Gap 8.3 and 9.4, were obtained from the ATCC and FITC-conjugated HLE-1 was purchased from Becton-Dickinson (MountainView, CA). Additionalexperiments were performed using a commercial anti-lck antiserum purchased from Upstate Biotechnology (Lake Placid, NY) and ananti-lck antiserum generously provided by R.Abraham (Rochester, MN) with results identical to those shown. Anti-fyn antiserum was purchased from Upstate Biotechnology or generously provided by R. Immunoprecipitation and Western Blotting-Immunoprecipitations were performed after cells were stimulated with the activating anti-TCR mAb,C305(1:500 dilution of ascites) or with medium alone. The cells were washed three times, lysed in the digitonin buffer, subjected to immunoprecipitation with MOPC 195 or mAb 9.4, SDS-PAGE and autoradiography.
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