title: The native structure of the activated Raf protein kinase is a membrane-bound multi-subunit complex.
abstract: Raf is a mitogen-stimulated protein kinase that functions as a component of the signaling cascade that leads to the stimulation of mitogen-activated protein kinase. Here we show that the native structure of Raf is a large multi-subunit protein complex with an apparent mass of 300-500 kDa that interacts with Ras and the mitogen-activated protein kinase kinase Mek. Analysis of the structure of the Raf complex demonstrates that it contains a single Raf protein kinase together with the molecular chaperones hsp90 and p50. The Raf-hsp90-p50 complex was observed in starved cells and in cells activated with serum or phorbol ester. Thus, changes in complex formation with hsp90 and p50 are not required for activation of the Raf protein kinase. However, Raf activation caused by Ras was associated with the translocation of the cytoplasmic Raf-hsp90-p50 complex to the cell membrane. Significantly, it is only the membrane-bound complex that exhibits increased protein kinase activity. Thus, the Ras-activated Raf protein kinase functions as a membrane-bound multi-subunit complex.
authors.affiliation: Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester 01605.
authors.forename: M
authors.initials: M
authors.lastname: Wartmann
authors.forename: R J
authors.initials: RJ
authors.lastname: Davis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D007106:Immune Sera; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
chemical_list: D007106:Immune Sera; D046911:Macromolecular Substances; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases