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J. Biol. Chem., Vol. 279, Issue 47, 48692-48701, November 19, 2004

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Src Tyrosine Kinase Inhibitor PP2 Markedly Enhances Ras-independent Activation of Raf-1 Protein Kinase by Phorbol Myristate Acetate and H₂O₂^*

Michael Lee, Ji-Young Kim, and Wayne B. Anderson¶

From the Laboratory of Genetic Toxicology, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Yusong, Daejeon 305-600, Korea and ^¶Laboratory of Cellular Oncology, NCI, National Institutes of Health, Bethesda, Maryland 20892-4263

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.

Received for publication, March 22, 2004 , and in revised form, August 30, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, P. O. Box 123, Yusong, Daejeon, 305-600, South Korea. Tel.: 82-42-610-8031; Fax: 82-42-610-8172; E-mail: mikelee{at}kitox.re.kr.

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