Abstract
The bases that support the versatility of the T cell receptor (TCR) to generate distinct T cell responses remain unclear. We have previously shown that mutant cells in the transmembrane domain of TCRbeta chain are impaired in TCR-induced apoptosis but are not affected in other functions. Here we describe the biochemical mechanisms by which this mutant receptor supports some T cell responses but fails to induce apoptosis. Extracellular signal-regulated protein kinase (ERK) is activated at higher and more sustained levels in TCRbeta-mutated than in wild type cells. Conversely, activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase is severely reduced in mutant cells. By attempting to link this unbalanced induction to altered upstream events, we found that ZAP-70 is normally activated. However, although SLP-76 phosphorylation is normally induced, TCR engagement of mutant cells results in lower tyrosine phosphorylation of LAT but in higher tyrosine phosphorylation of Vav than in wild type cells. The results suggest that an altered signaling cascade leading to an imbalance in mitogen-activated protein kinase activities is involved in the selective impairment of apoptosis in these mutant cells. Furthermore, they also provide new insights in the contribution of TCR to decipher the signals that mediate apoptosis distinctly from proliferation.
Highlights
The bases that support the versatility of the T cell receptor (TCR) to generate distinct T cell responses remain unclear
Impaired in Vivo Tyrosine Phosphorylation of LAT in TCR Chain Mutant Cells—We have previously shown that T cell clones bearing a single replacement in the ITAM-like motif of the TCR chain transmembrane domain express high amounts of TCR-CD3 complexes, similar to those of wild type cells
Immunoprecipitation with specific antiLAT antibodies followed by immunoblot with anti-phosphotyrosine showed that in comparison with wild type cells, TCRinducible tyrosine phosphorylation of LAT was clearly diminished in mutant cells (Fig. 1A)
Summary
Wild type and mutant clones have been previously described [29, 30]. They were obtained by reconstitution of the TCR-negative Jurkat variant 31.13 with either a wild type V3 TCR cDNA or with a mutant cDNA that contains a tyrosine to leucine mutation in the C-terminal tyrosine of the transmembrane domain. No clonal variation was observed between different clones derived from independent wild type or mutant cDNA transfections [29, 30]. The cells were maintained in RPMI supplemented with 10% fetal calf serum and antibiotics. Expression of TCR-CD3 complex was routinely tested by flow cytometry before each assay
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