Tyrosine Phosphorylation of the Proto-Oncoprotein Raf-1 Is Regulated by Raf-1 Itself and the Phosphatase Cdc25A

Abstract

There is a growing body of evidence demonstrating that Raf-1 is phosphorylated on tyrosines upon stimulation of a variety of receptors. Although detection of Raf-1 tyrosine phosphorylation has remained elusive, genetic analyses have demonstrated it to be important for Raf-1 activation. Here we report new findings which indicate that Raf-1 tyrosine phosphorylation is regulated in vivo. In both a mammalian and baculovirus expression system, a kinase-inactive allele of Raf-1 was found to be tyrosine phosphorylated at levels much greater than that of wild-type Raf-1. The level of tyrosine phosphate on Raf-1 was markedly increased upon treatment with phosphatase inhibitors either before or after cell lysis. Cdc25A was found to dephosphorylate Raf-1 on tyrosines that resulted in a significant decrease in Raf-1 kinase activity. In NIH 3T3 cells, coexpression of wild-type Raf-1 and phosphatase-inactive Cdc25A led to a marked increase in Raf-1 tyrosine phosphorylation in response to platelet-derived growth factor. These data suggest that the tyrosine phosphorylation of Raf-1 is regulated not only by itself but also by Cdc25A.

ACKNOWLEDGMENTS

We thank Helen Piwnica-Worms for providing plasmids encoding various forms of Cdc25A and baculoviruses encoding Cdc25A and Cdc25C; Anjana Rao for providing plasmid pLGP3, encoding HA-tagged wild-type Raf-1; Zhimin Zhu and Stephan Muhlebach for helpful technical advice; and Helen Piwnica-Worms, Kathy Campbell, Fred King, Joanne Chan, and Ibrahim Aksoy for critical reading of the manuscript.

This work was supported by NIH grants 2RO1CA43803-11A1 (to T.M.R.) and CA49152 (to B.G.N.).