Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane.
A conserved tyrosine kinase-activated signal transduction pathway has recently been identified that comprises the plasma membrane-bound small guanine-nucleotide-binding protein Ras and the protein kinases Raf, MAP-kinase kinase and MAP kinase. GTP-bound Ras interacts directly with the amino-terminal regulatory domain of Raf, but although Ras and Raf can be coimmunoprecipitated from ligand-stimulated cells, Ras-GTP does not stimulate the kinase activity of Raf in vitro. Furthermore, we have failed to detect Ras in preparations of active detergent-solubilized Raf, demonstrating that once it is activated, Raf does not require Ras. Whereas Raf is normally cytosolic, in cells expressing active Ras, Raf is associated with the plasma membrane. This led us to investigate whether Ras is required to localize Raf to the plasma membrane in order for Raf to become activated. We fused the membrane localization signal of K-Ras(4B) to the carboxy terminus of Raf. This protein is constitutively active and can be further activated by epidermal growth factor, independently of Ras. Our results indicate that Ras functions as a regulated, membrane-bound anchor for Raf, and that other signal(s) also contribute to Raf activation.
Leevers
S J
SJ
Section of Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, London, UK.
Paterson
H F
HF
Marshall
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D016716:PC12 Cells; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D014162:Transfection
D021382:Protein Sorting Signals; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D006160:Guanosine Triphosphate; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras)