Regulation of RAF activity by 14-3-3 proteins: RAF kinases associate functionally with both homo- and heterodimeric forms of 14-3-3 proteins.
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.
Fischer
Andreas
A
Bayerisches Krebsforschungszentrum, University of Wuerzburg, 97078 Wuerzburg, Germany.
Baljuls
Angela
A
Bayerisches Krebsforschungszentrum, University of Wuerzburg, 97078 Wuerzburg, Germany.
Reinders
Joerg
J
Institute of Functional Genomics, University of Regensburg, 93053 Regensburg, Germany.
Nekhoroshkova
Elena
E
Bayerisches Krebsforschungszentrum, University of Wuerzburg, 97078 Wuerzburg, Germany.
Sibilski
Claudia
C
Bayerisches Krebsforschungszentrum, University of Wuerzburg, 97078 Wuerzburg, Germany.
Metz
Renate
R
Bayerisches Krebsforschungszentrum, University of Wuerzburg, 97078 Wuerzburg, Germany.
Albert
Stefan
S
Bayerisches Krebsforschungszentrum, University of Wuerzburg, 97078 Wuerzburg, Germany.
Rajalingam
Krishnaraj
K
Emmy Noether Group of the Deutsche Forschungsgemeinschaft, Institute for Biochemistry II, Goethe University Medical School, 60596 Frankfurt, Germany.
Hekman
Mirko
M
Bayerisches Krebsforschungszentrum, University of Wuerzburg, 97078 Wuerzburg, Germany.
Rapp
Ulf R
UR
Bayerisches Krebsforschungszentrum, University of Wuerzburg, 97078 Wuerzburg, Germany. Electronic address: rappur@mail.uni-wuerzburg.de.
D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D017931:DNA Primers; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D053719:Tandem Mass Spectrometry; D048490:raf Kinases / Q000378:metabolism*
D048948:14-3-3 Proteins; D017931:DNA Primers; D020033:Protein Isoforms; D048490:raf Kinases