Impaired Binding of 14-3-3 to C-RAF in Noonan Syndrome Suggests New Approaches in Diseases with Increased Ras Signaling

Abstract

The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser²⁵⁹, a regulatory site for inhibition by 14-3-3 proteins. We show that these mutations impair binding of 14-3-3 proteins to C-RAF and alter its subcellular localization by promoting Ras-mediated plasma membrane recruitment of C-RAF. By presenting biophysical binding data, the 14-3-3/C-RAFpS²⁵⁹ crystal structure, and cellular analyses, we indicate a mechanistic link between a well-described human developmental disorder and the impairment of a 14-3-3/target protein interaction. As a broader implication of these findings, modulating the C-RAFSer²⁵⁹/14-3-3 protein-protein interaction with a stabilizing small molecule may yield a novel potential approach for treatment of diseases resulting from an overactive Ras-RAF-MAPK pathway.

This work was supported by DFG grants SFB 642 (to C.O.) and SFB 487 (Project C3 [to A.B. and U.R.R.]), DFG grant OT 414/1-1 (to P.T.), BMBF grant GO-Bio 0313873 (to M.W., R.R., and M.R.) and a Ph.D. scholarship from the IMPRS (to M.M.).

We thank Mirko Hekman for his helpful support regarding biosensor measurements. We appreciate the work done by the Dortmund Protein Facility (DPF). We thank Alfred Wittinghofer for helpful discussions and the staff at the Swiss Light Source, beamline X10SA, for support during crystallographic data collection.