Src tyrosine kinase inhibitor PP2 induces a sustained Ras-independent Activation of Raf-1 Protein Kinase by PMA 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. In the present study, we have demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-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 while 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 (PDGF)-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.