title: Allosteric activation of functionally asymmetric RAF kinase dimers.
abstract: Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, Box 8118, St. Louis, MO 63110, USA; Howard Hughes Medical Institute, Washington University School of Medicine, 660 South Euclid, Box 8118, St. Louis, MO 63110, USA.
authors.forename: Jiancheng
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, Box 8118, St. Louis, MO 63110, USA.
authors.forename: Edward C
authors.initials: EC
authors.lastname: Stites
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, Box 8118, St. Louis, MO 63110, USA.
authors.forename: Haiyang
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, Box 8118, St. Louis, MO 63110, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Germino
authors.affiliation: Department of Chemistry/Biochemistry, University of California San Diego, 9500 Gilman Drive, Mail code 0654, La Jolla, CA 92093, USA; Department of Pharmacology, University of California San Diego, 9500 Gilman Drive, Mail code 0654, La Jolla, CA 92093, USA.
authors.forename: Hiruy S
authors.initials: HS
authors.lastname: Meharena
authors.affiliation: Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.
authors.forename: Philip J S
authors.initials: PJS
authors.lastname: Stork
authors.affiliation: Department of Chemistry/Biochemistry, University of California San Diego, 9500 Gilman Drive, Mail code 0654, La Jolla, CA 92093, USA; Department of Pharmacology, University of California San Diego, 9500 Gilman Drive, Mail code 0654, La Jolla, CA 92093, USA; Howard Hughes Medical Institute, University of California San Diego, 9500 Gilman Drive, Mail code 0654, La Jolla, CA 92093, USA.
authors.forename: Alexandr P
authors.initials: AP
authors.lastname: Kornev
authors.affiliation: Department of Chemistry/Biochemistry, University of California San Diego, 9500 Gilman Drive, Mail code 0654, La Jolla, CA 92093, USA; Department of Pharmacology, University of California San Diego, 9500 Gilman Drive, Mail code 0654, La Jolla, CA 92093, USA; Howard Hughes Medical Institute, University of California San Diego, 9500 Gilman Drive, Mail code 0654, La Jolla, CA 92093, USA. Electronic address: staylor@ucsd.edu.
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, Box 8118, St. Louis, MO 63110, USA; Howard Hughes Medical Institute, Washington University School of Medicine, 660 South Euclid, Box 8118, St. Louis, MO 63110, USA. Electronic address: ashaw@wustl.edu.
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Shaw
mesh_terms: D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D019281:Dimerization; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014364:Tryptophan / Q000378:metabolism; D048490:raf Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014364:Tryptophan; D011494:Protein Kinases; C097199:KSR-1 protein kinase; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D048490:raf Kinases