AP-1 dependent signal transduction in murine T cell clones

TCR engagement in the absence of co-stimulatory signaling induces T cell clonal anergy. We have previously shown that activation of MAPK and the formation and transactivation of AP-1 transcriptional factor complexes were down-regulated in anergic T cells. Currently, we are developing a transient co-transfection system to examine how defective MAPK activities directly cause this reduced AP-1 function. GAL4 DNA binding domains have been fused with the transactivation domain of individual Jun and Fos proteins including c-Jun, JunD, junB, and c-Fos. The fusion constructs were then co-transfected with a Luciferase reporter gene driven by the GAL4 DNA binding element. Luciferase activity in co-trransfected anergic T cells. High levels of GAL4 fusion protein expression could be detected in Jurkat cells. Additionally, significantly increased activity for all of the AP-1 transactivation domains except that of junB were demonstrated in Jurkat cells after PMA and ionomycin stimulation. The fusion constructs and the reporter plasmid are now being transiently transfected into resting murine A.E7 T cells by electroporation. Thus, these constructs will allow us to exam whether their activation is affected by clonal anergy.