Discovery of Raf Family Is a Milestone in Deciphering the Ras-Mediated Intracellular Signaling Pathway

Abstract

The Ras-Raf-MEK-ERK signaling pathway, the first well-established MAPK pathway, plays essential roles in cell proliferation, survival, differentiation and development. It is activated in over 40% of human cancers owing to mutations of Ras, membrane receptor tyrosine kinases and other oncogenes. The Raf family consists of three isoforms, A-Raf, B-Raf and C-Raf. Since the first discovery of a truncated mutant of C-Raf as a transforming oncogene carried by a murine retrovirus, forty years of extensive studies have provided a wealth of information on the mechanisms underlying the activation, regulation and biological functions of the Raf family. However, the mechanisms by which activation of A-Raf and C-Raf is accomplished are still not completely understood. In contrast, B-Raf can be easily activated by binding of Ras-GTP, followed by cis-autophosphorylation of the activation loop, which accounts for the fact that this isoform is frequently mutated in many cancers, especially melanoma. The identification of oncogenic B-Raf mutations has led to accelerated drug development that targets Raf signaling in cancer. However, the effort has not proved as effective as anticipated, inasmuch as the mechanism of Raf activation involves multiple steps, factors and phosphorylation of different sites, as well as complex interactions between Raf isoforms. In this review, we will focus on the physiological complexity of the regulation of Raf kinases and their connection to the ERK phosphorylation cascade and then discuss the role of Raf in tumorigenesis and the clinical application of Raf inhibitors in the treatment of cancer.

Keywords: ERK; MEK; Raf; growth control; oncogenes; phosphorylation; signal transduction.

Figure 1

Structure of Raf family kinases. All Raf isoforms comprise three conserved regions: conserved region 1 (CR1) contains a Ras-binding domain (RBD) and a Cys-rich domain (CRD); conserved region 2 (CR2) is characterized by a Ser/Thr-rich sequence where 14-3-3 binds and inhibits Raf; conserved region 3 (CR3) is the kinase domain where the B-Raf V600E mutation is found in cancer. At the C-terminus, the second site promotes dimerization via binding to 14-3-3. BRS is a B-Raf-specific site. The viral oncoproteins v-Raf and v-mil have amino-terminal truncations and are fused with the N-myristoylated (N-myr) viral Gag protein. Four conserved phosphorylation sites of each Raf isoform are indicated in rectangles, including 14-3-3 binding sites and phoshorylattion sites in the activation loop. NtA: N-terminal acidic region.

Figure 2

The Ras/Raf/MEK/ERK signaling cascade. Growth factor binds to receptor tyrosine kinase and activates growth-factor receptor-bound 2 (GRB2) and Son of Sevenless (SOS) to load GTP to Ras. Then, Ras-GTP recruits Raf to the plasma membrane where Raf is activated, leading to sequential phosphorylation and activation of MEK and ERK. Activated ERK then phosphorylates a variety of substrates and elicits various cellular responses. RBD-CRD-CR3 designates essential domains of Raf and CR3 is the kinase domain.

Figure 3

Functional classes of B-Raf mutations. Class I B-Raf mutants contain V600E/D mutations in the activation loop which can signal as active monomers, independent of Ras. Class II B-Raf mutants are Ras-independent and signal as dimers. Class III B-Raf mutants have reduced kinase activity and drive the activation of ERK signaling by transactivating wild-type Raf which signals as mutant B-Raf–wild-type C-Raf dimers. These mutants require active Ras to trigger a signaling cascade.

Figure 4

Conformation transition of the Raf kinase domain. The kinase domain consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe) linked through a hinge. αC-helix and DFG (green) alter from OUT to IN position upon Raf activation, resulting in a dimer with side-to-side interface “closed” conformation. The conformational transition between inactive and active states is shown.

Figure 5

Functional properties of different RAF inhibitors. The upper part shows the effect of RAF inhibitors on monomeric RAF kinases; the lower part shows the effect of RAF inhibitors on dimeric RAF kinases. The first and second generations of Raf inhibitors lead to paradoxical activation in dimeric kinases. The inhibitor binds to one protomer within an RAF dimer, causing conformational change and decreasing the affinity of the inhibitor for the other protomer, as well as substantial transactivation of this protomer (dotted arrow), resulting in higher downstream signaling activation. The third generations of Pan-Raf inhibitors bind to monomeric and dimeric kinases with similar affinity. The third generation of paradox breakers disrupt the B-Raf dimer interface and specifically inhibit B-Raf dimerization but not C-Raf homodimerization, although they bind to C-Raf. INH: inhibitor. RAF designates any isoforms of Raf.