14-3-3 facilitates Ras-dependent Raf-1 activation in vitro and in vivo.
14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. We show here that 14-3-3 is bound to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol. A model that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation is proposed.
Roy
S
S
Queensland Cancer Fund Laboratory of Experimental Oncology, Department of Pathology, University of Queensland Medical School, Brisbane 4006, Australia.
McPherson
R A
RA
Apolloni
A
A
Yan
J
J
Lane
A
A
Clyde-Smith
J
J
Hancock
J F
JF
D048948:14-3-3 Proteins; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D011506:Proteins; D004815:Epidermal Growth Factor; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D015689:Oncogene Protein p21(ras)