B-Raf and C-Raf are required for Ras-stimulated p42 MAP kinase activation in Xenopus egg extracts

Abstract

During mitosis, a select pool of MEK1 and p42/p44 MAPK becomes activated at the kinetochores and spindle poles, without substantial activation of the bulk of the cytoplasmic p42/p44 MAPK. Recently, we set out to identify the MAP kinase kinase kinase (MAPKKK) responsible for this mitotic activation, using cyclin-treated Xenopus egg extracts as a model system, and presented evidence that Mos was the relevant MAPKKK . However, a second MAPKKK distinct from Mos was readily detectable as well. Here, we partially purify this second MAPKKK and identify it as B-Raf. No changes in the activity of B-Raf were detectable during progesterone-induced oocyte maturation, after egg fertilization, or during the early embryonic cell cycle, arguing against a role for B-Raf in the mitotic activation of MEK1 and p42 MAPK. Ras proteins can bring about activation of MEK1 and p42 MAPK in extracts, and Ras may contribute to signaling from the classical progesterone receptor during oocyte maturation and from receptor tyrosine kinases during early embryogenesis. We found that both B-Raf and C-Raf, but not Mos, are required for Ras-induced MEK1 and p42 MAPK activation. These data indicate that two upstream stimuli, active Ras and active Cdc2, utilize different MAPKKKs to activate MEK1 and p42 MAPK. © 2006 Nature Publishing Group All rights reserved.