Mechanistic principles of RAF kinase signaling

Abstract

The RAF family of kinases are key components acting downstream of receptor tyrosine kinases and cells employ several distinct mechanisms to strictly control their activity. RAF transitions from an inactive state, where the N-terminal regulatory region binds intramolecularly to the C-terminal kinase domain, to an open state capable of executing the phosphoryl transfer reaction. This transition involves changes both within and between the protein domains in RAF. Many different proteins regulate the transition between inactive and active states of RAF, including RAS and KSR, which are arguably the two most prominent regulators of RAF function. Recent developments have added several new twists to our understanding of RAF regulation. Among others, dimerization of the RAF kinase domain is emerging as a crucial step in the RAF activation process. The multitude of regulatory protein-protein interactions involving RAF remains a largely untapped area for therapeutic applications.

Fig. 1

The domain organization of RAF (a) and KSR (b) proteins. Conserved regions (CR) and conserved areas (CA) are indicated at the top of each panel, as is the negative charge region (N) in the RAF proteins. Conserved phosphorylation sites are indicated underneath each isoform. Phosphorylation sites known or suspected of mediating binding to 14-3-3 proteins are colored red (negative regulation) or green (positive regulation). The size of each isoform is noted at the right. D-RAF and D-KSR are the Drosophila paralogs and Ce denotes C. elegans proteins. All other proteins depicted are from human. S309 and S404 in human KSR1 are the equivalent of S297 and S392 in mouse KSR1 (see text). The unique region of KSR2 is represented as a shaded box. CRD Cysteine-rich domain, P proline-rich, RBD RAS-binding domain, S/T serine/threonine-rich

Fig. 2

An allosteric mechanism for activation of the kinase domain of RAF. RAF exists in an inactive conformation characterized by the monomeric state of its kinase domain. This monomeric state represents a kinase domain isolated from neighbouring kinase domains, but maintains other regulatory interactions that stabilize the inactive state as described in the text (for simplicity, only the C-terminal kinase domain of RAF is diagrammed; the N- and C-terminal lobes of the kinase domain are indicated). Upon RAS activation, the kinase domain of RAF transitions from an inactive monomer to an active side-to-side dimer state characterized structurally by the juxtaposition of a critical helix (helix αC) in the N-lobe that is thought to lock the kinase domain in a productive conformation competent for catalytic activity. A ribbons representation of the side-to-side dimer is shown (based on PDB ID 1UWH; ref. [137]). The cellular pool of RAF/KSR proteins in higher-order metazoa comprises multiple isoforms and the mechanism by which the cell selectively drives dimer formation between two given isoforms remains unknown