Leucine zipper domain of 52 kDa SS-A/Ro promotes protein dimer formation and inhibits in vitro transcription activity

摘要

Two forms of the human 52 kDa SS-A/Ro protein autoantigen, 52α and 52β, are products of alternative mRNA splicing. The 52α form is ubiquitously expressed whereas 52β, lacking the central leucine zipper domain, has been detected at higher levels than 52α during certain stages of fetal development. Because 52α has sequence similarity with macromolecules associated with transcriptional regulation and the two forms differ only in the 52β does not contain the leucine zipper, their roles in protein dimer formation and in transcriptional activity were examined. Employing the yeast two-hybrid system, 52α was shown to interact with itself but not 52β. The homodimerization of 52α was independently confirmed in gel filtration chromatography using in vitro cDNA template derived translation products and in HL-60 cell extracts; two peaks were observed corresponding to dimer and monomer of 52α, while in vitro the translation product of 52β exhibited only a single monomer peak. In addition, dimer formation was also demonstrated in a chemical cross-linking experiment using HeLa cells transfected with 52α. To evaluate effects on transcription, eukaryotic expression plasmids encoding 52α or 52β fused with the GAL4 DNA binding (DB) domain were co-transfected into 293 cells together with a luciferase reporter vector. A 6-fold increase in transcription activity of the reported was detected with the GAL4-DB-52β fusion constructs compared to GAL4-DB-52α or the empty vector control. We speculate that the ratio of cellular 52α and 52β may play in important role in regulating gene expression as potential repressor and activator respectively.