Fyn and Lck tyrosine kinases regulate tyrosine phosphorylation of p105CasL, a member of the p130Cas docking protein family, in T-cell receptor-mediated signalling

Abstract

We have previously shown that engagement of the T-cell receptor (TCR)/CD3 complex with anti-CD3 antibody induces tyrosine phosphorylation of p105CasL (CasL), a member of the p130Cas docking protein family. In the present work, we attempted to determine which protein tyrosine kinases (PTKs) regulate TCR-mediated phosphorylation of CasL. We show here that an association between CasL and two types of Src family PTKs, Fyn and Lck, is induced by anti-CD3 cross-linking of human H9 T cells. In contrast, ZAP-70, another PTK that also plays a critical role in the TCR signalling, failed to bind CasL, even after anti-CD3 stimulation. In vitro kinase assays revealed that Fyn and Lck, but not ZAP-70, were capable of phosphorylating CasL. Moreover, we found that CasL was constitutively hyperphosphorylated in vivo in splenocytes of MRL-MP-lpr/lpr mice, in which overproduction and excessive activation of Fyn and Lck have previously been shown to occur. Constitutive in vivo binding of CasL to both kinases was also demonstrated in lpr splenocytes. These results strongly suggest that CasL is a substrate for Fyn and Lck PTKs in TCR signal transduction.

Figure 1

Anti-CD3 cross-linking induces tyrosine phosphorylation of CasL. (a) H9 T cells were immunoprecipitated with U71 (lane 1) and Cas2 (lane 2), and were immunoblotted with clone 21. (b) H9 T cells were immunoprecipitated with U71 before (lane 1) and after (lane 2) anti-CD3 cross-linking, followed by antiphosphotyrosine immunoblotting (upper panel). This membrane was reprobed with clone 21 to verify that the same amount of protein was loaded into each lane.

Figure 3

Fyn and Lck, but not ZAP-70, kinases phosphorylate CasL in vitro. CasL, Fyn, Lck and ZAP-70 were separately immunoprecipitated from cell extracts of unstimulated H9 T cells. The CasL immunoprecipitate was divided into four: CasL immunoprecipitate alone as a control (lane 1); or mixed with anti-Lck (lane 2), anti-Fyn (lane 3) or anti-ZAP-70 (lane 4) immunoprecipitates. Each mixture was subjected to an in vitro kinase assay in the presence of ATP. CasL was reprecipitated with U71 and subjected to antiphosphotyrosine immunoblotting (upper panel), as detailed in the Materials and methods. The same membrane was reprobed with clone 21 (lower panel). IP, immunoprecipitate.

Figure 2

Anti-CD3 cross-linking induces association of CasL with Fyn and Lck kinases but not with ZAP-70. Cell extracts from H9 T cells, either unstimulated (lanes 1, 3 and 5) or stimulated (lanes 2, 4 and 6) with anti-CD3 cross-linking, were immunoprecipitated with antibodies against ZAP-70 (lanes 1 and 2), Lck (lanes 3 and 4) and Fyn (lanes 5 and 6). The immunoprecipitates were subjected to immunoblotting with antiphosphotyrosine antibody. The arrow indicates the 105 000 MW protein whose tyrosine phosphorylation was enhanced in response to anti-CD3 cross-linking and is associated with Lck and Fyn but not with ZAP-70. The upper part of the membrane (>90 000 MW) was reprobed with clone 21 (middle panel), while the lower part (<90 000 MW) was reprobed with antibodies against ZAP-70 (lane 1 and 2), Lck (lanes 3 and 4) and Fyn (lanes 5 and 6). IP, immunoprecipitate.

Figure 4

CasL expressed by lpr splenocytes was highly tyrosine phosphorylated and was associated with Lck and Fyn kinases. (a) Splenocytes obtained from 20-week-old lpr and +/+ mice were immunoprecipitated with U71, followed by antiphosphotyrosine immunoblotting. The same membrane was reprobed with clone 21 (anti-Cas). (b) Splenocyte extracts from 20-week-old lpr and +/+ mice were immunoprecipitated with anti-Lck, and probed with clone 21 immunoblotting. The same membrane was reprobed with anti-Lck. (c) Splenocyte extracts from 20-week-old lpr and +/+ mice were immunoprecipitated with anti-Fyn, and probed with clone 21 immunoblotting. The same membrane was reprobed with anti-Fyn. IP, immunoprecipitate.