Deciphering Signaling Pathways In Vivo: The Ras/Raf/Mek/Erk Cascade
The Ras/Raf/MEK/ERK cascade is a highly conserved signal transduction module, whose activation results in a number of different physiological outcomes. Depending on the cell type or the stimulus used, the pathway has been implicated in proliferation, diff
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1. Introduction The ERK-MAPK pathway was the first signal transduction cascade to be discovered and described from the cell membrane to the nucleus. The pathway has been intensely studied for about 20 years and is the paradigm for MAP kinase modules in general. Downstream of membrane receptors, the small G protein Ras recruits Raf from the cytosol to the cell membrane, where activation occurs (1). Activated Raf binds and phosphorylates MEK, which, in turn, activates ERK. An impressive roster of ERK substrates has been described, comprising membrane and cytoskeletal proteins, cytosolic enzymes, and transcription factors (2). The pathway is activated in 70% of all human cancers (3) and in a number of congenital progressing conditions (4). Clearly, the enzymes of the Raf/MEK/ ERK cascade are prime candidates for molecule-targeted therapies.
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_26, © Springer Science+Business Media, LLC 2010
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Galabova-Kovacs and Baccarini
Yet, little is known about their role in the whole organism. We use conventional and conditional ablation to identify the essential biological functions of Raf and Mek. To understand how the ERK pathway is wired in vivo, we combine phenotype and signaling analysis in the context of the whole organism (in vivo) with biochemical experiments in ablated cells. Using this combination, we could show that B-Raf is the nonredundant activator of the Erk pathway in vivo in the developing mouse placenta and in a mouse tumor model of insulinoma, in which cases it promotes angiogenesis (5, 6), and in the central nervous system, where it regulates the differentiation of dorsal root ganglia (7) and oligodendrocytes (8). C-Raf is crucial for Erk activation only in erythroid differentiation (9), but its essential roles in survival (10–12), migration, and tumorigenesis (13, 14) are independent of its enzymatic activity and are due to its ability to bind to, and inhibit, other serine/threonine kinases. Finally, we have discovered an unexpected essential role of MEK1 in downregulating Mek-2/ Erk signaling in the context of a Mek1:Mek2 heterodimer, establishing Mek1 as the critical modulator of Mek/Erk signaling (15). The methods used to define the signaling pathways affected by the ablation of Raf or Mek in vivo rely essentially on the availability of (phospho) specific antibodies, reflecting the activation state of individual pathways. Used in immunohistochemistry or immunofluorescence, and combined with antibodies defining cells of a specific lineage, these antibodies are a powerful tool for pathway analysis in vivo.
2. Materials 2.1. Immunohistochemistry and Immunofluorescence
1. PBS, phosphate buffer solution: 0.2 g KCl, 8 g NaCl, 1.4 g Na2HPO4, 0.2 g KHPO4 in 1 L ddH2O, adjusted to pH 7.4 with HCl, store at 4°C.
2.1.1. Fixation
2. 4% (w/v) Paraformaldehyde (PFA) in PBS, adjust to pH 7.4, store at 4°C. 3. 70% Ethanol.
2.1.2. Dehydration and Paraffin Embedding Accordi
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