Ras-Mediated Activation of the Raf Family Kinases

Abstract

The extracellular signal-regulated kinase (ERK) cascade comprised of the Raf, MEK, and ERK protein kinases constitutes a key effector cascade used by the Ras GTPases to relay signals regulating cell growth, survival, proliferation, and differentiation. Of the ERK cascade components, the regulation of the Raf kinases is by far the most complex, involving changes in subcellular localization, protein and lipid interactions, as well as alterations in the Raf phosphorylation state. The Raf kinases interact directly with active, membrane-localized Ras, and this interaction is often the first step in the Raf activation process, which ultimately results in ERK activation and the downstream phosphorylation of cellular targets that will specify a particular biological response. Here, we will examine our current understanding of how Ras promotes Raf activation, focusing on the molecular mechanisms that contribute to the Raf activation/inactivation cycle.

Figure 1.

Raf domain structure and protein interactions. (A) The Raf kinases can be divided into two functional domains: an amino-terminal regulatory domain and a carboxy-terminal kinase domain and they contain three conserved regions (CRs): CR1, which contains a Ras-binding domain (RBD) and a cysteine-rich domain (CRD); CR2, an area rich in serine/threonine residues (S/T); and CR3, which contains the protein kinase domain. (B) Numerous Raf-binding partners have been identified.

Figure 2.

Regulation of the Raf kinases by phosphorylation. (Top) Raf is both positively and negatively regulated by phosphorylation on numerous sites. (Bottom) Shown are the amino acid sequence alignments of the sites of Raf phosphorylation, with the residues phosphorylated shown in red, and the relevant kinases indicated above the sequences. A glutamic acid residue that can act as a phosphomimetic to promote the active conformation of the activation segment (AS) is also shown in green.

Figure 3.

Regulation of the Raf kinase domain by dimerization. Shown are the crystal structures of a monomeric, B-Raf kinase domain in an inactive conformation (PDB, 4WO5) and of dimerized B-Raf kinase domains in an active conformation (taken from the B-Raf–MEK complex PDB, 4MNE). In the monomeric, inactive state, the αC-helix is shifted outward and residues adjacent to the DFG motif in the activation segment (AS) form a small helix known as the AS-H1-helix. When Raf kinase domains dimerize in a side-to-side manner, the αC-helix shifts inward to the active position and the AS-H1-helix is disrupted, allowing residues in the R-spine to align. The conserved Raf dimer interface motif RKTR is shown in red, the R-spine residues in the αC-helix (L505) and in the DFG motif (D595) are indicated in yellow, and the sequences comprising the AS-H1-helix are shown in green.

Figure 4.

The Raf activation–inactivation cycle. A model depicting the Raf kinases in a presignaling inactive state, a signaling active state, and a postsignaling inactive state is shown (see text for details). GDP, Guanosine diphosphate; GTP, guanosine triphosphate; RBD, Ras-binding domain; CRD, cysteine-rich domain; ERK, extracellular signal-regulated kinase.