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          Institute: MPI für Herz- und Lungenforschung (W. G. Kerckhoff Institut)     Collection: Publikationen des W. G. Kerckhoff-Instituts     Display Documents



ID: 474540.0, MPI für Herz- und Lungenforschung (W. G. Kerckhoff Institut) / Publikationen des W. G. Kerckhoff-Instituts
Regulation of RAF activity by 14-3-3 proteins: RAF kinases associate functionally with both homo- and heterodimeric forms of 14-3-3 proteins
Authors:Fischer, A.; Baljuls, A.; Reinders, J.; Nekhoroshkova, E.; Sibilski, C.; Metz, R.; Albert, S.; Rajalingam, K.; Hekman, M.; Rapp, U. R.
Date of Publication (YYYY-MM-DD):2009
Title of Journal:J Biol Chem
Volume:284
Issue / Number:5
Start Page:3183
End Page:94
Audience:Not Specified
Abstract / Description:Mammalian 14-3-3 proteins play a crucial role in the activation process of RAF kinases. However, little is known about the selectivity of the mammalian 14-3-3 isoforms with respect to RAF association and activation. Using mass spectrometry, we analyzed the composition of the 14-3-3 isoforms attached to RAF kinases and found that B-RAF associates in vivo with 14-3-3 at much higher diversity than A- and C-RAF. We also examined in vitro binding of purified mammalian 14-3-3 proteins to RAF kinases using surface plasmon resonance techniques. While B- and C-RAF exhibited binding to all seven 14-3-3 isoforms, A-RAF bound with considerably lower affinities to epsilon, tau, and sigma 14-3-3. These findings indicate that 14-3-3 proteins associate with RAF isoforms in a pronounced isoform-specific manner. Because 14-3-3 proteins appear in dimeric forms, we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. For that purpose, the budding yeast Saccharomyces cerevisiae, possessing only two 14-3-3 isoforms (BMH1 and BMH2), served as testing system. By deletion of the single BMH2 gene, we found that both homo- and heterodimeric forms of 14-3-3 can participate in RAF activation. Furthermore, we show that A-, B-, and C-RAF activity is differentially regulated by its C-terminal and internal 14-3-3 binding domain. Finally, prohibitin, a scaffold protein that affects C-RAF activation in a stimulatory manner, proved to interfere with the internal 14-3-3 binding site in C-RAF. Together, our results shed more light on the complex mechanism of RAF activation, particularly with respect to activation steps that are mediated by 14-3-3 proteins and prohibitin.
Free Keywords:14-3-3 Proteins/chemistry/metabolism/*physiology; Base Sequence; Binding Sites; Biosensing Techniques; DNA Primers; Dimerization; Electrophoresis, Polyacrylamide Gel; Hela Cells; Humans; Immunoprecipitation; Protein Isoforms/chemistry/metabolism/*physiology; Tandem Mass Spectrometry; raf Kinases/*metabolism
External Publication Status:published
Document Type:Article
Communicated by:N. N.
Affiliations:MPI für physiologische und klinische Forschung
External Affiliations:Bayerisches Krebsforschungszentrum, University of Wuerzburg, Wuerzburg, Germany.
Identifiers:ISSN:0021-9258 (Print) 0021-9258 (Linking)
URL:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=..
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