14-3-3 Antagonizes Ras-Mediated Raf-1 Recruitment to the Plasma Membrane To Maintain Signaling Fidelity

Abstract

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,G37Ras, ^V23TC21, and ^V38R-Ras. Disruption of 14-3-3 binding at CR2 did not recover binding of Raf-1 to ^V12,G37Ras but allowed more efficient recruitment of Raf-1 to the plasma membrane and stimulated its phosphorylation on S338. Finally, ^V12Ras, but not ^V12,G37Ras, 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.

We thank C. J. Marshall, D. Barford, and members of the Signal Transduction Team for their helpful comments on the work in progress and for comments on the manuscript.

This work was funded by Cancer Research UK and The Institute of Cancer Research.