14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity.
We have investigated the role that S259 phosphorylation, S621 phosphorylation, and 14-3-3 binding play in regulating Raf-1 activity. We show that 14-3-3 binding, rather than Raf-1 phosphorylation, is required for the correct regulation of kinase activity. Phosphorylation of S621 is not required for activity, but 14-3-3 binding is essential. When 14-3-3 binding to conserved region 2 (CR2) was disrupted, Raf-1 basal kinase activity was elevated and it could be further activated by (V12,G37)Ras, (V23)TC21, and (V38)R-Ras. Disruption of 14-3-3 binding at CR2 did not recover binding of Raf-1 to (V12,G37)Ras but allowed more efficient recruitment of Raf-1 to the plasma membrane and stimulated its phosphorylation on S338. Finally, (V12)Ras, but not (V12,G37)Ras, displaced 14-3-3 from full-length Raf-1 and the Raf-1 bound to Ras. GTP was still phosphorylated on S259. Our data suggest that stable association of Raf-1 with the plasma membrane requires Ras-mediated displacement of 14-3-3 from CR2. Small G proteins that cannot displace 14-3-3 fail to recruit Raf-1 to the membrane efficiently and so fail to stimulate kinase activity.
Light
Yvonne
Y
Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
Paterson
Hugh
H
Marais
Richard
R
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
D048948:14-3-3 Proteins; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins