A novel role for Cdk1/cyclin B in regulating B-raf activation at mitosis.
MAPK activity is important during mitosis for spindle assembly and maintenance of the spindle checkpoint arrest. We previously identified B-Raf as a critical activator of the MAPK cascade during mitosis in Xenopus egg extracts and showed that B-Raf activation is regulated in an M-phase-dependent manner. The mechanism that mediates B-Raf activation at mitosis has not been elucidated. Interestingly, activation of 95-kDa B-Raf at mitosis does not require phosphorylation of Thr-599 and Ser-602 residues (Thr-633 and Ser-636 in Xenopus B-Raf), previously shown to be essential for B-Raf activation by Ras. Instead, we provide evidence for Cdk1/cyclin B in mediating mitotic activation of B-Raf. In particular, Cdk1/cyclin B complexes associate with B-Raf at mitosis in Xenopus egg extracts and contribute to its phosphorylation. Mutagenesis and in vitro kinase assays demonstrated that Cdk1/cyclin B directly phosphorylates B-Raf at Serine-144, which is part of a conserved Cdk1 preferential consensus site (S(144)PQK). Importantly, phosphorylation of Ser-144 is absolutely required for mitotic activation of B-Raf and subsequent activation of the MAPK cascade. However, substitution of a phospho-mimicking amino acid at Ser-144 failed to produce a constitutive active B-Raf indicating that, in addition of Ser-144 phosphorylation, other regulatory events may be needed to activate B-Raf at mitosis. Taken together, our data reveal a novel cell cycle mechanism for activating the B-Raf/MEK/MAPK cascade.
Borysov
Sergiy I
SI
Molecular Oncology Program, H. Lee Moffitt Cancer and Research Institute, Tampa, FL 33612, USA.
Guadagno
Thomas M
TM
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D019926:Cyclin B / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D020935:MAP Kinase Signaling System; D008938:Mitosis; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D014981:Xenopus
D019926:Cyclin B; D004791:Enzyme Inhibitors; D012694:Serine; D048493:Proto-Oncogene Proteins B-raf; D016203:CDC2 Protein Kinase