title: B-Raf is critical for MAPK activation during mitosis and is regulated in an M phase-dependent manner in Xenopus egg extracts.
abstract: Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. Here we purified and characterized the MEK kinase activity present in Xenopus M phase-arrested egg extracts. Our results show that B-Raf was the critical MEK kinase required for M phase activation of the MAPK pathway. Consistent with this, B-Raf was activated and underwent hyperphosphorylation in an M phase-dependent manner. Interestingly B-Raf hyperphosphorylation at mitosis occurred, at least in part, as a consequence of a feedback loop involving MAPK-mediated phosphorylation within a conserved C-terminal SPKTP motif. The kinase activity of a B-Raf mutant defective at both phosphorylation sites was substantially greater than its wild type counterpart when incubated in Xenopus M phase egg extracts. Furthermore suppression of MAPK feedback at mitosis enhanced B-Raf activity, whereas constitutive activation of MAPK at mitosis strongly suppressed B-Raf activity. These results suggest that feedback phosphorylation by MAPK negatively regulates B-Raf activity at mitosis. Collectively our data demonstrate for the first time a role for B-Raf at mitosis and provide new insight into understanding the regulation and function of B-Raf during cell proliferation.
authors.affiliation: Molecular Oncology Program, Department of Interdisciplinary Oncology, H. Lee Moffitt Comprehensive Cancer Center and Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida 33612, USA.
authors.forename: Sergiy I
authors.initials: SI
authors.lastname: Borysov
authors.forename: Anthony W M
authors.initials: AW
authors.lastname: Cheng
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Guadagno
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D008938:Mitosis; D008954:Models, Biological; D008969:Molecular Sequence Data; D048493:Proto-Oncogene Proteins B-raf / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
chemical_list: D048493:Proto-Oncogene Proteins B-raf; D020930:MAP Kinase Kinase Kinases