Deciphering signaling pathways in vivo - the Ras/Raf/Mek/Erk cascade

The Ras/Raf/MEK/ERK pathway represents the oldest paradigm of a cytosolic signal transduction cascade, and its constitutive activation as a result of mutations is considered a key event in the development of several human malignancies and developmental disorders. The components of the pathway, particularly the Raf kinases A-, B-, and C-Raf (Raf-1), are considered attractive therapeutic targets, but surprisingly little is known about their essential functions in the context of the whole organism. The research goal of the Baccarini lab is to define the essential function of C-Raf, B-Raf and their target Mek-1 in in vivo models of tissue development, remodeling, and neoplasia. To this end, they combine phenotype analysis in the context of the whole organism (in vivo) as well as of cells (ex vivo) with biochemical experiments in ablated cells to elucidate the molecular basis of a given phenotype. In the past few years, the Baccarini lab has shown that B-Raf is the non-redundant activator of the Erk pathway in vivo in the developing mouse placenta (Figures 1 and 2) and in a mouse tumor model of insulinoma, in which cases it promotes angiogenesis (1,2), and in the central nervous system, where it regulates the differentiation of dorsal root ganglia (3) and of oligodendrocytes (4). C-Raf is crucial for Erk activation only in erythroid differentiation (5). In contrast, C-Raf is essential to promote survival and migration independently of its enzymatic activity as a MEK kinase (6). Instead, C-Raf binds to and negatively regulates two proapoptotic kinases [Mst2 (7,8) and Ask1 (9)] and the Rho-dependent kinase Rok-alpha, which controls cell shape, migration, and the expression of the death receptor Fas (10,11). Most recently, we could demonstrate that oncogenic Ras promotes the interaction between C-Raf and Rok-alpha, and that the negative regulation of Rok-alpha by C-Raf is absolutely necessary for both development and maintenance of Ras-induced epidermal tumors (12). In the absence of C-Raf, Rok-alpha hyperactivity induces the rapid differentiation of these tumors, which disappear never to relapse (12). Mechanistically, Rok-alpha inhibition is mediated by the autoinhibitory domain of C-Raf, which is structurally similar to the autoinhibitory domain of Rok-alpha. Upon activation, both C-Raf and Rok-alpha are converted from a "closed" conformation, in which their autoinhibitory domains contact and inhibit the respective kinase domains, into an "open" conformation, in which the kinase domains can accept and phosphorylate substrates. In this situation, the autoinhibitory domain of C-Raf, like an ill-fitting lego brick, contacts the kinase domain of Rok-alpha, restraining its activity (14). 

By showing that their essential in vivo functions are fundamentally different, these results have changed the way we look at Raf kinases and have opened new possibilities for molecule-targeted therapy.

More recently, the group has discovered an unexpected essential role of MEK1 in downregulating Mek-2/Erk signaling. MEK1 mediates the regulation of MEK2 in the context of a Mek1:Mek2 heterodimer negatively regulated by Erk-mediated phosphorylation of Mek1. These data establish Mek1 as the critical modulator of Mek/Erk signaling in vivo and in vitro (13).

The Raf/MEK/ERK pathway
Erk signaling and pathway cross-talk mediated by the Raf kinases ->= activation, -I = repression.

Current projects

The lab will continue to focus on deciphering signaling pathways in vivo. We carry out studies in different cell lineages, given the often-contrasting evidence about the role and relevance of signal transduction pathways studied in different cellular systems. For each process, we are using several in vitro models and at least one in vivo model, to ensure the relevance and the validity of the information obtained.
 
Manuela Baccarini is the coordinator of the PhD Program “Molecular Mechanisms of Cell Signaling” (W1220), funded by the Austrian National Scientific Research Fund.

We offer competitive PhD projects in the areas of development, tumorigenesis, inflammation, and biogenesis of signaling complexes.
 
Our work is currently supported by the SFB 021 of the Austrian National Scientific Research Fund by the Obermann-Mahlke Foundation, and by grants Health-F2-2009-223151 (INFLA-CARE) and LSH-CT-2006-037731 (GROWTHSTOP) of the European Community

References

1.  Galabova-Kovacs, G. et al. Essential role of B-Raf in ERK activation during extraembryonic development. Proc Natl Acad Sci U S A 103, 1325-30 (2006).
2.  Sobczak, I. et al. B-Raf is required for ERK activation and tumor progression in a mouse model of pancreatic beta-cell carcinogenesis. Oncogene 27, 4779-87 (2008).
3.  Zhong, J. et al. Raf kinase signaling functions in sensory neuron differentiation and axon growth in vivo. Nat Neurosci 10, 598-607 (2007).
4.  Galabova-Kovacs, G. et al. Essential role of B-Raf in oligodendrocyte maturation and myelination during postnatal central nervous system development. J Cell Biol 180, 947-55 (2008).
5.  Rubiolo, C. et al. A balance between Raf-1 and Fas expression sets the pace of erythroid differentiation. Blood 108, 152-9 (2006).
6.  Mikula, M. et al. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf- 1 gene. Embo J 20, 1952-1962. (2001).
7.  Matallanas, D. et al. RASSF1A elicits apoptosis through an MST2 pathway directing proapoptotic transcription by the p73 tumor suppressor protein. Mol Cell 27, 962-75 (2007). 8.  O'Neill, E., Rushworth, L., Baccarini, M. & Kolch, W. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306, 2267-70 (2004).
9.  Yamaguchi, O. et al. Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J Clin Invest 114, 937-43 (2004).
10.  Ehrenreiter, K. et al. Raf-1 regulates Rho signaling and cell migration. J Cell Biol 168, 955-64 (2005).
11.  Piazzolla, D., Meissl, K., Kucerova, L., Rubiolo, C. & Baccarini, M. Raf-1 sets the threshold of Fas sensitivity by modulating Rok-{alpha} signaling. J. Cell Biol. 171, 1013-1022 (2005).
12.  Ehrenreiter, K., Kern, F., Velamoor, V., Meissl, K., Galabova-Kovacs, G., Sibilia, M., and Baccarini, M. Raf-1 addiction in Ras-induced carcinogenesis. Cancer Cell, 16, 149-160 (2009).
13. Niault, T, Sobczak, I., Meissl, K., Weitsman, G., Piazzolla, D., Maurer, G., Kern, F., Ehrenreiter, K., Hamerl, M., Moarefi, I., Leung, T., Carugo, O., Ng, T., and Baccarini, M. From autoinhibition to inhibition in trans: the Raf-1 regulatory domain inhibits Rok-alpha kinase activity. J Cell Biol, 187, 335-342 (2009)
14.  Catalanotti, F. et al. A MEK1-MEK2 heterodimer determines the strength and duration of the ERK signal. Nat Struct Mol Biol 16, 294-303 (2009).