Characterization of Raf-1 activation in mitosis

We have used site-directed mutagenesis to explore the mechanisms underlying Raf-1 activation in mitosis, and we have excluded most previously characte rized activating interactions. Our results indicate that the primary locus of activation lies in the carboxyl-half of the molecule, although the exten t of activation can be influenced by the amino-proximal region, particularl y by the Raf-1 zinc finger, We also found that Raf-1 is hyperphosphorylated in mitosis at multiple sites within residues 283-302 and that these hyperp hosphorylations are not required for activation, In addition, neither Mek1 nor Mek2 are stably activated in coordination with Raf-1 in nocodazole-arre sted cells. Overall, the data suggest that the mechanism(s) responsible for activating Raf-1 during mitosis, and the subsequent downstream effects, ar e distinct from those involved in growth factor stimulation.