title: Src tyrosine kinase inhibitor PP2 markedly enhances Ras-independent activation of Raf-1 protein kinase by phorbol myristate acetate and H2O2.
abstract: Recently we reported that simultaneous treatment of NIH 3T3 cells with the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H2O2) resulted in synergistic activation of Raf-1 kinase (Lee, M., Petrovics, G., and Anderson, W. B. (2003) Biochem. Biophys. Res. Commun. 311, 1026-1033). In this study we have demonstrated that PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, greatly potentiated the ability of PMA and/or H2O2 to activate Raf-1 kinase, whereas it blocked the tyrosine phosphorylation of Raf-1. Unlike PMA/H2O2 treatment, which showed transient activation, PP2-mediated Raf-1 activation was sustained and continued to increase through 4 h of treatment. Transient transfection studies with a dominant-negative mutant of Ras (N19Ras) indicated that this PP2-induced activation of Raf-1 was Ras-independent. Moreover, PP2 showed no effect on platelet-derived growth factor-induced Raf-1 activation. Interestingly, mutation of the reported Raf-1 Src family tyrosine kinase phosphorylation site by conversion of tyrosines 340 and 341 to phenylalanine (YY340/341FF Raf) had limited effect on the ability of PP2 to induce significant stimulation of Raf-1 kinase activity. Taken together, our results suggest that a tyrosine phosphorylation event is involved in the negative feedback regulation of Raf-1. Inhibition of a Src family tyrosine kinase by PP2 appears to alleviate this tyrosine kinase-mediated inhibition of Raf-1 and allow activating modification(s) of Raf-1 to proceed. This PP2 effect resulted in significant and sustained Ras-independent activation of Raf-1 by PMA and H2O2.
authors.affiliation: Laboratory of Genetic Toxicology, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Yusong, Daejeon 305-600, Korea. mikelee@kitox.re.kr
authors.forename: Michael
authors.initials: M
authors.lastname: Lee
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Kim
authors.forename: Wayne B
authors.initials: WB
authors.lastname: Anderson
mesh_terms: D000818:Animals; D001665:Binding Sites; D002461:Cell Line, Transformed; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D000939:Epitopes / Q000737:chemistry; D005799:Genes, Dominant; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D008954:Models, Biological; D041681:NIH 3T3 Cells; D010649:Phenylalanine / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
chemical_list: C412373:AG 1879; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D000939:Epitopes; D011743:Pyrimidines; D014443:Tyrosine; D010649:Phenylalanine; D006861:Hydrogen Peroxide; D019908:Proto-Oncogene Proteins c-raf; D013755:Tetradecanoylphorbol Acetate