Novel C-Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation.
The C-Raf kinase is regulated by numerous phosphorylation steps. To quantify the most prominent phosphorylation sites of C-Raf, we performed mass spectrometry analysis of wild-type C-Raf and the constitutively active C-Raf mutant C-Raf-Y340D/Y341D. We confirmed phosphorylation of most of the sites reported in the literature with the exception that we did not detect phosphorylation of threonine 268/269 (autophosphorylation sites) and threonine 491/serine 494 (kinase activation loop). Importantly, we detected novel phosphorylation sites at the positions of serine 296 and 301. The degree of phosphorylation in these positions depends on the level of activation of C-Raf. Furthermore, we show here, using point mutant forms of C-Raf kinases with serine to alanine and serine to aspartic acid substitution, that serines 296 and 301 contribute to negative regulation of C-Raf.
Hekman
Mirko
M
Institut fuer Medizinische Strahlenkunde und Zellforschung, University of Wuerzburg, 97078 Wuerzburg, Germany.
Fischer
Andreas
A
Wennogle
Lawrence P
LP
Wang
Y Karen
YK
Campbell
Sharon L
SL
Rapp
Ulf R
UR
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics; D002522:Chlorocebus aethiops; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010449:Peptide Mapping; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics / Q000378:metabolism*
D012694:Serine; D019908:Proto-Oncogene Proteins c-raf