Sandbox Reserved 707: Difference between revisions

== Regulation<ref>PMID:22569528</ref> ==

[[Image:The RAS RAF MEK ERK pathway.jpg|left|400px|thumb| Diagram of the RAS-RAF-MEK-ERK pathway]]

RAF kinases participate in the RAS-RAF-MEK-ERK signal transduction cascade, which is sometimes denoted as the mitogen-activated protein kinase (MAPK) cascade<ref>PMID:12471243</ref>. Growth factors bind to receptor tyrosine kinases (RTKs), resulting in RAS activation. RAF proteins are one of a family of effector proteins activated by RAS, and they in turn stimulate the activation of MEK, which subsequently stimulates ERK activity. ERK phosphorylates both cytosolic and nuclear proteins, thereby mediating the cellular responses cells make when this pathway is activated.<br />

Under non stimulatory conditions a serine of CR2 domain and another near the C-terminus are phosphorylated and are bound to 14-3-3 domain. To activate RAF Kinases, RAS-GTP has to interact with the RDB Domain. This is necessary but not enough to activate the RAF Kinase.

One characteristic that distinguish B-RAF from A or C-RAF is the presence of Asp448 and Asp449 on the N-terminal region, which bear negative charges. Phosphorylation of <scene name='Sandbox_Reserved_707/Psite_of_activation_segment/1'>Thr599</scene> and Ser602 in the activation segment is essential for B-RAF activation. That’s why the basal activity of B-RAF is greater than A or C-RAF.<br />

Although X-ray structures of B-RAF kinase domains were said to be monomeric, each of the six structures in the protein data bank (2009), including B-RAF (V600E), showed that the asymmetric unit of the crystal contains two kinase domains that interact in a unique side-to-side fashion involving the N-lobe<ref>PMID:19727074</ref>. This side-to-side dimerization involves the αC helix, an important determinant of active and inactive conformations. Based upon ultracentrifugation studies, it is demonstrated that the catalytic domain of human B-RAF forms homodimers. Arg509 of B-RAF occurs in the side-to-side interface, and these investigators found that the Arg509<ref>PMID:19727074</ref> His mutant exists as a monomer. But B-RAF can form heterodimers too<ref>PMID:16508002</ref>. Ser621 and residues in the αC helix of C-RAF participate in dimer formation. B-RAF–C-RAF heterodimers are more active than either homodimer. Phosphorylation of B-RAF at Thr753 as catalyzed by ERK destabilizes heterodimer formation with C-RAF and decreases kinase activity.

In a first study Heidorn and al. blocked N-RAS and C-RAF by RNA interference. The result showed that the ERK activity  was blocked, indicating that RAS and C-RAF participate in the paradoxical response. They reported that 885-A binding to B-RAF or mutation to a kinase-dead B-RAF drives their binding to C-RAF. The introduction of a gatekeeper mutation in B-RAF abolishes the ability of B-RAF inhibitors to induce the binding of B-RAF to C-RAF. Inhibitor binding to B-RAF in the presence of activated RAS induces B-RAF binding to C-RAF leading to C-RAF activation and increased downstream ERK phosphorylation and activation.<br />

In a second study, Poulikakos and al. reported that six ATP-competitive RAF inhibitors induce ERK activation in cells with activated RAS and wild-type B-RAF but inhibit signaling in activated mutant B-RAF cells<ref>PMID:20179705</ref>. B-RAF and C-RAF form homo- and heterodimers following RAS activation. PLX4032 and PLX4720, RAF kinase inhibitors, induce the phosphorylation of MEK and ERK in wild-type and B-RAF -/- mouse embryonic fibroblasts. The response is diminished in C-RAF -/- fibroblasts, arguing on the importance of C-RAF in paradoxical MEK-ERK activation. <br />

In a third study, Hatzivassiliou and colleagues reported that RAF inhibitor treatment results in the paradoxical induction of phospho-MEK and phospho-ERK levels in the wild-type RAS/RAF human Melanoma<ref>PMID:20141835</ref>. They showed that knockdown of C-RAF, but not B-RAF, in human colorectal carcinoma

RAS mutations occur in 15–30% of all human cancers, and B-RAF mutations occur in 30–60% of melanomas, 30–50% of thyroid cancers, and 5–20% of colorectal cancers. B-RAF mutants occur in a variety of cancers while mutants of the other two RAF enzymes in cancers are very rare. The majority of B-RAF mutations occur in the activation segment or in the glycine-rich loop. These mutations disrupt the inactive state to favor the active state. A Val600Glu mutation is one of the more known B-RAF mutations. This mutation occurs within the activation segment where the introduction of negative charges favors the formation of an active conformation. The introduction of glutamate into the activation segment of C-RAF fails to produce an activated enzyme most likely owing to the need for a negatively charged N-region for activity. <br />