Reifung funktioneller Membranmikrodomänen zur Raf-abhängigen Signaltransduktion entlang des membrangebundenen sekretorischen Weges
Final Report Abstract
RAF kinases, although known for more than 20 years, are crucial players in regulation of many cellular processes in response to mitogenic signals. Although all three mammalian RAF kinases share similarities in their overall structure, many differences in their regulation and physiological function have been identified by a multitude of approaches. We employed the advantages of yeast system, which does not contain endogenous RAF kinases, in combination with biochemical methods to establish the role of phosphorylation at 14-3-3 protein-binding sites and interaction with different 14-3-3 proteins in regulation of RAF kinases. There are two phosphorylated 14-3-3 binding sites in the structure of RAF kinases. A proximal site located in the N-terminal part of proteins (serine 259 in C-RAF) and site distal (serine 621 in C-RAF) located in C-terminal part of molecules. We found that the RAF isoforms are differently dependent on phosphorylation of distal 14-3-3 binding site. A-RAF, the least characterized RAF remains mysterious in many aspects. It is differently phosphorylated, uniquely localized and A-RAF knockout mice have comparatively mild phenotype. Here we studied the role of this kinase in the regulation of membrane trafficking. C-terminally truncated ARI 49 was identified as a deletion mutant with a specific localization and lethal phenotype in yeasts. In mammals, AR149/DA-RAF2 also exhibits a specific localization on tubular endosomes and seems to act in down-regulation of endosome-localized A-RAF signaling. Results on tissues expressing DA-RAF2 and its role in cell differentiation published by others suggest that the function of AR149 may go far beyond endocytosis. The novel findings on the A-RAF localization and the interaction with ARF6 have led us to propose a new model of A-RAF function and open new perspectives for investigation of role of A-RAF in nervous system. Our findings on A- RAF regulation of endocytic recycling could explain the most severe phenotypes of A-RAF knockout animals, such as ataxia, rigidity of the musculature and continuous tremor. Efficient endocytosis of synaptic vesicles, their reloading with neutrotransmitters and subsequent recycling are essential for the proper function of neuronal tissues. Depletion of A-RAF, which, as we found out, affects this process may disunite this machinery, leading to the phenotypes above.
Publications
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(2006) BuCy Rafs drive cells into MEK addiction. Cancer Cell, 9: 9-12
Rapp, U.R., Götz, R. & Albert, S.
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(2006) Reversible membrane interaction of BAD requires two C-terminal lipid binding domains in conjunction with 14-3-3 protein binding. Journal of Biological Chemistry, 281 (25): 17321 -17336
Hekman, M., Albert, S., Galmiche, A., Rennefahrt, U.E.E., Fueller, J., Fischer, A., Puehringer, D., Wiese, S., & Rapp, U.R.
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(2006) Survival regulation by Ras and Raf. In: Apoptosis and Cancer Therapy, K.-M. Debatin and S. Fulda eds. WILEY-VCH
Drexler, H.C.A., Galmiche, A., Hekman, M., Albert, S., & Rapp, U.R.
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(2007) RAS oncogenes and their downstream targets. Biochimica et Biophysica Acta 1773(8): 1177-1195
Rajalingam, K., Schreck, R., Rapp, U.R. & Albert, S.
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(2009) Regulation of RAF activity by 14-3-3 proteins - RAF kinases associate functionally with both homo- and heterodimeric forms of 14-3-3 proteins. Journal of Biological Chemistry, 284(5): 3183-3194
Fischer, A., Baljuls, A., Reinders, J., Nekhoroshkova, E., Sibilski, C., Metz, R., Albert, S., Rajalingam, K., Hekman, M., & Rapp, U.R.
