THE CA2-ACTIVATED, PHOSPHOPROTEIN PHOSPHATASE CALCINEURIN IS SUFFICIENT FOR POSITIVE TRANSCRIPTIONAL REGULATION OF THE MOUSE IL-4 GENE( CALMODULIN)

We have studied the TCR mediated signal transduction pathways involved in transcriptional regulation of the mouse IL-4 gene. The sequences e xtending from base pair -766 to + 63 of the IL-4 gene were inserted up stream of a luciferase indicator gene. Transcriptional activity was ob served when the construct, [pIL-4(- 766)], was transfected into either the IL-4 producing cell line, EL-4, or the IL-4 non-producing T cell hybridoma, 68-41, but not in the L929 fibroblast cell line. By analysi s of deletion mutants of pIL-4(- 766), we identified a transcriptional regulatory element that is tightly associated with a signal coming fr om the TCR and which controls inducible activation of the IL-4 promote r. By analysis of deletion mutants of pIL-4(- 766), this latter elemen t was found between base pairs - 147 to - 17. Electrophoretic mobility shift assays indicated that expression of a nuclear binding protein w ith binding sites between base pairs - 84 and - 55 could be induced. B y competition and mutation analysis, the binding motif of this protein was determined to be AAAATTTTCC. Stimulation with ionomycin alone was sufficient to induce activity in pIL-4(- 766). Cyclosporin A inhibite d both the IL-4 promoter activity and activation of the inducible nucl ear protein. Transient over-expression of a constitutively active form of the Ca2+/Calmodulin-regulated protein phosphatase, calcineurin was sufficient to cause activation of pIL-4(- 766) without any additional stimulus. These results indicate that the signaling requirements for activation of upstream positive regulatory elements of the IL-4 gene a re distinct from those of the IL-2 gene. Ca2+ Mobilization is sufficie nt to activate the IL-4 promoter, whereas IL-2 gene transcription requ ires both Ca2+ mobilization and protein kinase C activation.