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Activation of B‐Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601
Author(s) -
Zhang BaoHong,
Guan KunLiang
Publication year - 2000
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/19.20.5429
Subject(s) - biology , phosphorylation , kinase , conserved sequence , microbiology and biotechnology , genetics , biochemistry , peptide sequence , gene
The Raf kinase family serves as a central intermediate to relay signals from Ras to ERK. The precise molecular mechanism for Raf activation is still not fully understood. Here we report that phosphorylation of Thr598 and Ser601, which lie between kinase subdomains VII and VIII, is essential for B‐Raf activation by Ras. Substitution of these residues by alanine (B‐RafAA) abolished Ras‐induced B‐Raf activation without altering the association of B‐Raf with other signaling proteins. Phosphopeptide mapping and immunoblotting with phospho‐specific antibodies confirmed that Thr598 and Ser601 are in vivo phosphorylation sites induced by Ras. Furthermore, replacement of these two sites by acidic residues (B‐RafED) renders B‐Raf constitutively active. Con sistent with these data, B‐RafAA and B‐RafED exhibited diminished and enhanced ability, respectively, to stimulate ERK activation and Elk‐dependent transcription. Moreover, functional studies revealed that B‐RafED was able to promote NIH 3T3 cell transformation and PC12 cell differentiation. Since Thr598 and Ser601 are conserved in all Raf family members from Caenorhabditis elegans to mammals, we propose that phosphorylation of these two residues may be a general mechanism for Raf activation.