The kinases of the Raf family have been intensively studied as activators of the mitogen-activated protein kinase kinase͞extra-cellular signal-regulated kinase (ERK) module in regulated and deregulated proliferation. Genetic evidence that Raf is required for ERK activation in vivo has been obtained in lower organisms, which express only one Raf kinase, but was hitherto lacking in mammals, which express more than one Raf kinase. Ablation of the two best studied Raf kinases, B-Raf and Raf-1, is lethal at midgestation in mice, hampering the detailed study of the essential functions of these proteins. Here, we have combined conventional and conditional gene ablation to show that B-Raf is essential for ERK activation and for vascular development in the placenta. B-Rafdeficient placentae show complete absence of phosphorylated ERK and strongly reduced HIF-1␣ and VEGF levels, whereas all these parameters are normal in Raf-1-deficient placentae. In addition, neither ERK phosphorylation nor development are affected in B-raf-deficient embryos that are born alive obtained by epiblastrestricted gene inactivation. The data demonstrate that B-Raf plays a nonredundant role in ERK activation during extraembyronic mammalian development in vivo.T he placenta is the first organ to develop during embryogenesis, and it supports the growth of the developing embryo by mediating the exchange of nutrients and wastes between the fetal and maternal circulatory systems. Placentation includes extensive angiogenesis, and reduced placental vascular development is associated with early embryonic mortality. Genetic studies have demonstrated a crucial role of VEGF, FGF, and their receptors in placental angiogenesis. In addition, the ablation of several signaling molecules operating downstream of receptor tyrosine kinases results in defects in placentation, often at the stage of labyrinth formation (1).The Raf kinases (A-Raf, B-Raf, and Raf-1) relay signals from tyrosine kinase receptors to the mitogen-activated protein kinase kinase (MEK)͞extracellular signal-regulated kinase (ERK) signaling module. Although most of the early work on the activation of the MEK͞ERK module was focused on Raf-1, evidence has accumulated that B-Raf is the main MEK kinase. Raf kinases from lower organisms (Caenorhabditis elegans lin-45 and Drosophila D-Raf ) are more similar to B-Raf than to the other two mammalian Raf kinases. Biochemical studies have indicated that B-Raf is the main MEK kinase found in fibroblast and brain lysates (2-5). Consistently, among the three Raf kinases, B-Raf binds best to MEK (6) and has the highest basal MEK kinase activity, both in vitro (7) and in fibroblasts, when expressed as a conditionally oncogenic form (8). Finally, B-Raf mutations resulting in increased MEK͞ERK activation have been discovered in a broad range of human tumors (9). All these observations hint at B-Raf as the archetypal mammalian MEK kinase, whereas Raf-1 and A-Raf have probably diverged to perform other functions. Growth-factor-stimulated ERK activation is reduced...
Mutations in the extracellular signal-regulated kinase (ERK) pathway, particularly in the mitogen-activated protein kinase/ERK kinase (MEK) activator B-Raf, are associated with human tumorigenesis and genetic disorders. Hence, B-Raf is a prime target for molecule-based therapies, and understanding its essential biological functions is crucial for their success. B-Raf is expressed preferentially in cells of neuronal origin. Here, we show that in mice, conditional ablation of B-Raf in neuronal precursors leads to severe dysmyelination, defective oligodendrocyte differentiation, and reduced ERK activation in brain. Both B-Raf ablation and chemical inhibition of MEK impair oligodendrocyte differentiation in vitro. In glial cell cultures, we find B-Raf in a complex with MEK, Raf-1, and kinase suppressor of Ras. In B-Raf–deficient cells, more Raf-1 is recruited to MEK, yet MEK/ERK phosphorylation is impaired. These data define B-Raf as the rate-limiting MEK/ERK activator in oligodendrocyte differentiation and myelination and have implications for the design and use of Raf inhibitors.
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