Inhibitory effects of rosmarinic acid on Lck SH2 domain binding to a synthetic phosphopeptide

Abstract

In the course of screening inhibitors from the methanol (MeOH) extracts of 168 medicinal plants against lymphocyte cell-specific kinase (Lck) Src-homology 2 (SH2) binding to a synthetic phosphotyrosine-containing peptide (phosphopeptide), we isolated rosmarinic acid from the MeOH extract of Prunella vulgaris, which showed specific inhibitory activity. The IC50 value for Lck SH2 binding to phosphopeptide (SGSGEEPQpYEEIPI) of hamster polyomavirus middle-sized tumor (hmT pY324) was 7 μM. However, even at concentrations of 0.1 to 1000 μM, no significant inhibitions were observed against other SH2 domains binding such as the growth factor receptor binding protein 2 (Grb2) SH2 domain to phosphopeptide of Shc and phospholipase Cγ1 (PLCγ1) SH2 domain to translational elongation factor 1α (EF1 ?) C-terminal. Rosmarinic acid inhibited interleukin-2 (IL-2) gene expression by 50% at a concentration of 8 μM in Jurkat cells stimulated with anti-CD3 and anti-CD4 antibodies. FK506 and cyclosporin A (CsA) employed as positive controls showed less than 30% inhibition at the same concentration. In addition, rosmarinic acid inhibited the intracellular [Ca2+]i increase in Jurkat cells after T cell activation in a dose-dependent manner at concentrations of 1.4 to 140 μM of rosmarinic acid, which is one of the earliest responses of antigen-specific T cell receptor (TCR) and of the upstream pathway of IL-2 expression. Taken together, these results suggest that rosmarinic acid has the potential to specifically inhibit Lck SH2 domain binding to its cognate ligand, including ZAP-70, Cb1, HS-1, and PLCγ1, and Lck-dependent Ca2+ signaling pathway of its downstream effector and finally to modulate IL-2 gene expression after T cell activation.