Fischer, Andreas and Baljuls, Angela and Reinders, Joerg and Nekhoroshkova, Elena and Sibilski, Claudia and Metz, Renate and Albert, Stefan and Rajalingam, Krishnaraj and Hekman, Mirko and Rapp, Ulf R. (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). pp. 3183-3194. ISSN 0021-9258, 1083-351X
Full text not available from this repository. (Request a copy)Abstract
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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | CYSTEINE-RICH DOMAIN; B-RAF; C-RAF; PHOSPHORYLATION SITES; IN-VIVO; A-RAF; POLYACRYLAMIDE-GELS; BIOLOGICAL-ACTIVITY; BINDING-SITES; CAUSE NOONAN; |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Institut für Funktionelle Genomik > Lehrstuhl für Funktionelle Genomik (Prof. Oefner) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 07 Oct 2020 08:33 |
| Last Modified: | 07 Oct 2020 08:33 |
| URI: | https://pred.uni-regensburg.de/id/eprint/29564 |
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