Inhibitory effects of rosmarinic acid on Lck SH2 domain binding to a synthetic phosphopeptide.
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 IC 50 value for Lck SH2 binding to phosphopeptide (SGSGEEPQpYEEIPI) of hamster polyomavirus middle-sized tumor (hmT pY324) was 7 microM. However, even at concentrations of 0.1 to 1000 microM, 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 Cgamma1 (PLCgamma1) SH2 domain to translational elongation factor 1alpha (EF1alpha) C-terminal. Rosmarinic acid inhibited interleukin-2 (IL-2) gene expression by 50 % at a concentration of 8 microM 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 [Ca 2+] i increase in Jurkat cells after T cell activation in a dose-dependent manner at concentrations of 1.4 to 140 microM 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 PLCgamma1, and Lck-dependent Ca 2+ signaling pathway of its downstream effector and finally to modulate IL-2 gene expression after T cell activation.
Ahn
Soon Cheol
SC
Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon, Korea.
Oh
Won Keun
WK
Kim
Bo Yeon
BY
Kang
Dae Ook
DO
Kim
Min Soo
MS
Heo
Gun Young
GY
Ahn
Jong Seog
JS
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D015704:CD4 Antigens / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D002934:Cinnamates / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D006224:Cricetinae; D053630:Depsides; D051380:GRB2 Adaptor Protein; D015870:Gene Expression; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D019169:Jurkat Cells; D007723:Korea; D008213:Lymphocyte Activation / Q000187:drug effects; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000138:chemical synthesis / Q000378:metabolism*; D010946:Plants, Medicinal / Q000737:chemistry; D011506:Proteins / Q000378:metabolism; D031347:Prunella / Q000737:chemistry*; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism; D018909:src Homology Domains / Q000187:drug effects*; D000097123:Rosmarinic Acid
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D017252:CD3 Complex; D015704:CD4 Antigens; D002118:Calcium; D002934:Cinnamates; D053630:Depsides; D051380:GRB2 Adaptor Protein; D007376:Interleukin-2; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051966:Phospholipase C gamma; D010750:Phosphoproteins; D011506:Proteins; D010738:Type C Phospholipases; D000097123:Rosmarinic Acid; C495948:GRB2 protein, human