title: Functional mapping of the N-terminal regulatory domain in the human Raf-1 protein kinase.
abstract: Raf-1 is a serine/threonine kinase poised at a key relay point in mitogenic signal transduction pathways from the cell surface to the nucleus. Activation of the transforming potential of Raf-1 has been associated with N-terminal truncation and/or fusion to other proteins, suggesting that the Raf-1 N-terminal half harbors a negative regulatory domain. Seven internal deletion mutants that together scan the entire N-terminal half of human Raf-1 protein were generated to map functional regions in this regulatory domain. Effects of the deletion mutations on kinase activity of Raf-1 were evaluated using a baculovirus/insect cell overexpression system and an in vitro kinase assay with the known physiological substrate of Raf-1, mitogen-activated protein kinase kinase. Deletion of amino acids 276-323 in the unique sequence between conserved regions 2 and 3 leads to modest elevation of Raf-1 basal kinase activity, whereas deletion of amino acids 133-180 in conserved region 1 results in diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal deletion mutants, including a truncated version that is transforming in rodent fibroblasts, exhibits greatly increased levels of basal kinase activity. In addition, while activation of Raf-1 kinase by Ras requires sequences in conserved region 1, only the C-terminal half containing the kinase domain of Raf-1 is required for activation by Src. These findings demonstrate that N-terminal deletions in Raf-1 do not necessarily result in constitutively elevated basal kinase activity and that the N-terminal regulatory domain is completely dispensable for Raf-1 activation by Src.
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109, USA.
authors.forename: Y H
authors.initials: YH
authors.lastname: Chow
authors.forename: K
authors.initials: K
authors.lastname: Pumiglia
authors.forename: T H
authors.initials: TH
authors.lastname: Jun
authors.forename: P
authors.initials: P
authors.lastname: Dent
authors.forename: T W
authors.initials: TW
authors.lastname: Sturgill
authors.forename: R
authors.initials: R
authors.lastname: Jove
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology; D011817:Rabbits; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D018631:ras Proteins / Q000502:physiology
chemical_list: D011518:Proto-Oncogene Proteins; D016392:Proto-Oncogene Proteins pp60(c-src); D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins