c-Raf

RAF proto-oncogene serine/threonine-protein kinase, also known as proto-oncogene c-RAF or simply c-Raf or even Raf-1, is an enzyme that in humans is encoded by the RAF1 gene. The c-Raf protein is part of the ERK1/2 pathway as a MAP kinase kinase kinase (MAP3K) that functions downstream of the Ras subfamily of membrane associated GTPases. C-Raf is a member of the Raf kinase family of serine/threonine-specific protein kinases, from the TKL (Tyrosine-kinase-like) group of kinases. The first Raf gene, v-Raf was found in 1983. It was isolated from the murine retrovirus bearing the number 3611. It was soon demonstrated to be capable to transform rodent fibroblasts to cancerous cell lines, so this gene was given the name Virus-induced Rapidly Accelerated Fibrosarcoma (V-RAF). A year later, another transforming gene was found in the avian retrovirus MH2, named v-Mil - that turned out to be highly similar to v-Raf. Researchers were able to demonstrate that these genes encode enzymes that have serine-threonine kinase activity. Normal cellular homologs of v-Raf and v-Mil were soon found in both the mouse and chicken genome (hence the name c-Raf for the normal cellular Raf gene), and it became clear that these too had a role in regulating growth and cell division. Now we know that c-Raf is a principal component of the first described mitogen-activated protein kinase (MAPK) pathway: ERK1/2 signaling. It acts as a MAP3 kinase, initiating the entire kinase cascade. Subsequent experiments showed that the normal, cellular Raf genes can also mutate to become oncogenes, by 'overdriving' MEK1/2 and ERK1/2 activity. In fact, vertebrate genomes contain multiple Raf genes. Several years later after the discovery of c-Raf, two further related kinases were described: A-Raf and B-Raf. The latter became the focus of research in recent years, since a large portion of human tumors carry oncogenic 'driver' mutations in the B-Raf gene. These mutations induce an uncontrolled, high activity of Raf enzymes. Thus diagnostic and therapeutic interest in Raf kinases reached a new peak in the recent years. The human c-Raf gene is located on chromosome 3. At least two isoforms of mRNA have been described (arising from inclusion or removal of an alternative exon) that display only minute differences. The shorter, major isoform - consisting of 17 exons - encodes a protein kinase of 648 amino acids. Similarly to many other MAPKKKs, c-Raf is a multidomain protein, with several additional domains to aid the regulation of its catalytic activity. On its N-terminal segment, a Ras-binding domain (RBD) and a C-kinase homologous domain 1 (C1 domain) are found next to each other. Structures of both conserved domains were solved in the past decades, shedding light on the mechanisms of their regulation. The Ras-binding domain displays a ubiquitin-like fold (like many other small G-protein associating domains) and selectively binds GTP-bound Ras proteins only. (You can see this interaction in high detail in the PDB box attached to the article. It shows Rap1 in complex with the RBD of c-Raf.) The C1 domain - immediately downstream of the Ras binding domain - is a special zinc finger, rich in cysteines and stabilized by two zinc ions. It is similar to the diacylglycerol-binding C1 domains of protein kinase C (PKC) enzymes. But unlike PKC, the C1 domains of Raf family kinases do not bind diacylglycerol. Instead, they interact with other lipids, such as ceramide or phosphatidic acid, and even aid in the recognition of activated Ras (GTP-Ras). The close proximity of these two domains as well as several lines of experimental data suggest that they act as a single unit to negatively regulate the activity of the protein kinase domain, by direct physical interaction. Historically, this autoinhibitory block was labelled as the CR1 region ('Conserved Region 1'), the hinge region being named CR2, and the kinase domain CR3. Unfortunately, the precise structure of the autoinhibited kinase remains unknown. Between the autoinhibitory domain block and the catalytic kinase domain, a long segment - characteristic to all Raf proteins - can be found. It is highly enriched in serine amino acids, but its precise sequence is poorly conserved across related Raf genes. This region appears to be intrinsically unstructured, and very flexible. Its most likely role is to act as a natural 'hinge' between the rigidly folded autoinhibitory and catalytic domains, enabling complex movements and profound conformational rearrangements within the molecule. This hinge region contains a small, conserved island of amino acids, that are responsible for 14-3-3 protein recognition, but only when a critical serine (Ser259 in human c-Raf) is phosphorylated. A second, similar motif is found on the extreme C-terminus (centered around the phosphorylatable Ser 621) of all Raf enzymes, but downstream of the kinase domain.