CONDITIONALLY ONCOGENIC FORMS OF THE A-RAF AND B-RAF PROTEIN-KINASES DISPLAY DIFFERENT BIOLOGICAL AND BIOCHEMICAL-PROPERTIES IN NIH 3T3 CELLS

The protein kinase domains of mouse A-Raf and B-Raf were expressed as fusion proteins with the hormone binding domain of the human estrogen receptor in mammalian cells. In the absence of estradiol, 3T3 and ratl a cells expressing Delta A-Raf:ER and Delta B-Raf:ER were nontransform ed, but upon the addition of estradiol the cells became oncogenically transformed. Morphological oncogenic transformation was more rapid and distinctive in cells expressing Delta B-Raf:ER compared with cells ex pressing Delta A-Raf:ER. Biochemical analysis of cells transformed by Delta-Raf:ER and Delta B-Raf:ER revealed several interesting differenc es. The activation of Delta B-Raf:ER consistently led to the rapid and robust activation of both MEK and p42/p44 MAP kinases. By contrast, t he activation of Delta A-Raf:ER led to a weak activation of MEK and th e p42/p44 MAP kinases. The extent of activation of MEK in cells correl ated with the ability of the different Raf kinases to phosphorylate an d activate MEK1 in vitro. Delta B-Raf:ER phosphorylated MEK1 approxima tely 10 times more efficiently than Delta Raf-1:ER and at least 500 ti mes more efficiently than Delta-Raf:ER under the conditions of the imm une-complex kinase assays. These results mere confirmed with epitope-t agged versions of the Raf kinase domains expressed in insect cells. Th e activation of all three Delta Raf:ER proteins in 3T3 cells led to th e hyperphosphorylation of the resident p74(raf-I) and mSOS1 proteins, suggesting the possibility of ''cross-talk'' between the different Raf kinases and feedback regulation of intracellular signaling pathways. The activation of either Delta B-Raf:ER or Delta Raf-1:ER in quiescent 3T3 cells was insufficient to promote the entry of the cells into DNA synthesis. By contrast, the activation of Delta A-Raf:ER in quiescent 3T3 cells was sufficient to promote the entry of the cells into S pha se after prolonged exposure to beta-estradiol. The Delta Raf:ER system has allowed us to reveal significant differences between the biologic al and biochemical properties of oncogenic forms of the Raf family of protein kinases. We anticipate that cells expressing these proteins an d other estradiol-regulated protein kinases will be useful tools in fu ture attempts to unravel the complex web of interactions involved in i ntracellular signal transduction pathways.