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 (EF1α) 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, Cbl, 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.

Prunella vulgaris - Labiatae - rosmarinic acid - Lck SH2 - Grb2 SH2 - PLC γ1 SH2 - IL-2 expression - intracellular [Ca²⁺]_i.

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