| Item type: | Article | ||||||||||||||||||||||||||||
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| Journal or Publication Title: | The Journal of biological chemistry | ||||||||||||||||||||||||||||
| Publisher: | AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC | ||||||||||||||||||||||||||||
| Place of Publication: | BETHESDA | ||||||||||||||||||||||||||||
| Volume: | 284 | ||||||||||||||||||||||||||||
| Number of Issue or Book Chapter: | 5 | ||||||||||||||||||||||||||||
| Page Range: | pp. 3183-3194 | ||||||||||||||||||||||||||||
| Date: | January 2009 | ||||||||||||||||||||||||||||
| Institutions: | Medicine > Institut für Funktionelle Genomik > Lehrstuhl für Funktionelle Genomik (Prof. Oefner) | ||||||||||||||||||||||||||||
| Identification Number: |
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| Classification: |
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| Keywords: | CYSTEINE-RICH DOMAIN; B-RAF; C-RAF; PHOSPHORYLATION SITES; IN-VIVO; A-RAF; POLYACRYLAMIDE-GELS; BIOLOGICAL-ACTIVITY; BINDING-SITES; CAUSE NOONAN; | ||||||||||||||||||||||||||||
| Dewey Decimal Classification: | 600 Technology > 610 Medical sciences Medicine 600 Technology > 610 Medical sciences Medicine | ||||||||||||||||||||||||||||
| Status: | Published | ||||||||||||||||||||||||||||
| Refereed: | Yes, this version has been refereed | ||||||||||||||||||||||||||||
| Created at the University of Regensburg: | Partially | ||||||||||||||||||||||||||||
| Item ID: | 30675 |
Web of ScienceAbstract
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 ...
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.
Metadata last modified: 29 Sep 2021 07:40
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