ORIGINAL ARTICLE B-Raf and C-Raf are required for Ras-stimulated p42 MAP kinase activation in Xenopus egg extracts J Yue, W Xiong 1 and JE Ferrell Jr Department of Molecular Pharmacology, Stanford University, Stanford, CA, USA 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. Oncogene (2006) 25, 3307–3315. doi:10.1038/sj.onc.1209354; published online 23 January 2006 Keywords: B-Raf; C-Raf; Mos; Ras; p42; MAPK; MEK1 Introduction ERK1 and ERK2 (extracellular signal-regulated kinases 1 and 2) are probably the best studied and best understood of the mitogen-activated protein kinases (MAPKs). ERK1 and ERK2 respond to diverse stimuli, and play important roles in cell proliferation, differ- entiation, and survival (Ferrell, 1996a; Robinson and Cobb, 1997; Lewis etal., 1998; Ferrell, 1999b; Widmann et al., 1999). Their immediate upstream activators are the kinases MEK1 and MEK2 (for MAPK kinase or ERK kinase), which phosphorylate ERK1 and ERK2 at a threonine and a tyrosine residue in the kinase domain activation loop. MEK1 and MEK2, in turn, are activated by phosphorylation of one or two serine sites in their activation loops (Alessi et al., 1994). Three families of MEK1/2-activating MAP kinase kinase kinases (MAPKKKs) have now been identified: the Raf family, consisting of the A-Raf, B-Raf, and Raf-1 or C-Raf proteins; the Ste11/Byr1-related MEKK family, consisting of MEKK1, MEKK2, MEKK3, and MEKK4; and the Mos oncoprotein. The diversity of the MAPKKKs may allow MEK1/2 and ERK1/2 proteins to plug into different upstream receptors and regulators. The serine/threonine protein kinases of the Raf family have long been implicated in relaying mito- genic signals to MAPKs (Morrison and Cutler, 1997; Kolch, 2000; Murakami and Morrison, 2001; Chong et al., 2003). For example, the gene for the best studied Raf protein, C-Raf, was initially identified as the cellular homolog of the retroviral oncogenes, v-raf and v-mil, underscoring the potential of C-Raf to be a mitogenic regulator (Jansen et al., 1984). More recently, B-Raf has been shown to be mutated in more than 60% of melanomas and a substantial percentage of many other human cancers (Davies et al., 2002; Pollock and Meltzer, 2002; Rajagopalan et al., 2002; Chong et al., 2003; Pollock et al., 2003; Wan et al., 2004), suggesting that this Raf family member is of particular importance in human disease. All three of the Raf proteins can be activated by Ras proteins, and all can phosphorylate and activate MEK1; C-Raf can activate MEK2 as well (Pritchard etal., 1995; Wu et al., 1996; Marais et al., 1997). The three Raf proteins are expressed in overlapping but distinct patterns, with A-Raf exhibiting the most restricted distribution. Likewise, studies of knockout mice suggest that there are both overlapping and isoform-specific functions of the three Raf proteins, with the B-Raf and C-Raf single knockouts producing embryonic lethality (showing that the Raf proteins are probably not completely redundant functionally), but with cells from the single knockouts showing relatively normal p42 MAPK responses (suggesting some redundancy between Received 15 September 2005; revised 25 October 2005; accepted 10 November 2005; published online 23 January 2006 Correspondence: Dr J Yue, Department of Molecular Pharmacology, Stanford University School of Medicine, CCSR Room 3160, 269 West Campus Drive, Stanford, CA 94305-5174, USA. E-mail: jyue@stanford.edu 1 Current address: W Xiong, The Scripps Research Institute, Mail Stop #SR202, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA. Oncogene (2006) 25, 3307–3315 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc