Function of Raf family kinases has been shown to play a role during organism development, cell cycle regulation, cell proliferation and differentiation, cell survival and apoptosis and many other cellular and physiological processes.
The RAF family of serine/threonine kinases are MAP3Ks that function downstream of the RAS family of small GTPases during mitogenic signal transduction. RAF was discovered by isolation from a murine sarcoma virus, in a manner remarkably similar to the discovery of Ras. In fact, RAF was the first oncogene discovered with Ser/Thr kinase activity. The RAF family of kinases includes ARAF, BRAF and CRAF/RAF-1.
Ras first recruits RAF to the plasma membrane, and then activates RAF by relieving autoinhibition mediated by its N-terminal regulatory domain. BRAF and CRAF form heterodimers that are more active than either of the monomers or homodimers. Since ARAF and CRAF require additional N-terminal phosphorylation to be fully activated while those sites on BRAF are constitutively phosphorylated, BRAF is the predominant RAF isoform that signals downstream of oncogenic Ras. In agreement with this, BRAF itself is frequently mutated in human tumors, while somatic mutations in ARAF or CRAF are rare. CRAF was the first of these proteins to be identified as the non-transforming (non-oncogenic) cellular homolog of the viral v-raf oncogene acquired by the murine retrovirus 3611-MSV. Each of these proteins share a common architecture, with three conserved regions (CR): CR1 and CR2 in the regulatory N-terminus and the C-terminal CR3 which encodes the kinase domain.
In quiescent cells, RAF exists in an auto-inhibited state, where the N-terminus suppresses the activity of the C-terminal catalytic domain. RAF activation begins with its recruitment to the inner leaflet of the plasma membrane by directly binding GTP-bound RAS via the RAS-binding domain (RBD) and forming secondary interactions via the cysteine rich domain (CRD), both of which are encoded within the CR1 region of the N-terminus. RAS binding and recruitment to the plasma membrane by itself is insufficient for the full activation of RAF. There are multiple phosphorylation events that are important to the regulation of RAF activity. Some of these events are inhibitory and require dephosphorylation by phosphatases to potentiate efficient activation.
For instance, N-terminal CRAF binding to RAS is shown to be sterically hindered by phosphorylation of Ser43, while phosphorylation of Ser233 and Ser259 permits binding of the 14-3-3 scaffold protein that, when bound, interferes with RAS binding and promotes the adoption of a closed, inactive conformation. Each of these sites is targeted by protein kinase A (PKA), and their inhibitory effects on CRAF activation can be overcome following dephosphorylation by PP2A. Other phosphorylation events are activating and occur following RAS-mediated recruitment to the plasma membrane. The phosphorylation of two residues within the N-region located at the amino terminus of CR3, Ser338 and Tyr341, is required for full activation of CRAF. The resultant negative charge in this region alleviates the intramolecular inhibition of the kinase domain by the N-terminus. While Src and Srcfamily kinases mediate the phosphorylation of Try341, the identity of the Ser338 kinase (or kinases) remains unclear. The p21 activated kinases (PAKs) have been implicated based on in vitro data, however, they fail to induce CRAF activation in a growth-factor dependent process. Two additional sites located within the kinase domain, Thr491 and Ser492, are essential for CRAF activation, however, the kinases that mediated their phosphorylation remain unknown; it is possible that these sites are subject to an auto-phosphorylating mechanism.
Reference: Eric Joseph. SMALL MOLECULE INHIBITION OF ACTIVATED MAP KINASE SIGNALING IN HUMAN CANCERS: BIOLOGICAL AND THERAPEUTIC IMPLICATIONS