B- and C-RAF Display Essential Differences in Their Binding to Ras

Abstract

Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD).Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B-and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.

Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia

“…However, it was shown recently that BRAF is frequently mutated in melanoma (27-70%), papillary thyroid cancer (36-53%), colorectal cancer (5-22%) and ovarian cancer (30%). 220,223,224 The reasons for mutation at BRAF and not RAF1 or ARAF in melanoma patients are not entirely clear. On the basis of mechanism of activation of BRAF, it may be easier to select for BRAF than either RAF1 or ARAF mutations.…”

“…Furthermore, B-Raf may be activated in the cytoplasm by non-farnesylated Ras, while Raf-1 requires farnesylated Ras for translocation to the cell membrane. 224 It was proposed recently that the structure of B-Raf, Raf-1 and A-Raf may dictate the ability of activating mutations to occur at these molecules, which can permit the selection of oncogenic forms. 221,224,225 These predictions have arisen from determining the crystal structure of B-Raf.…”

“…224 It was proposed recently that the structure of B-Raf, Raf-1 and A-Raf may dictate the ability of activating mutations to occur at these molecules, which can permit the selection of oncogenic forms. 221,224,225 These predictions have arisen from determining the crystal structure of B-Raf. 225 Like many enzymes, B-Raf is proposed to have small and large lobes, which are separated, by a catalytic cleft.…”

Regulation of RAF Activity by 14-3-3 Proteins

“…CR1 contains a Ras binding domain and a zinc binding domain, also called cysteine-rich domain. Although all RAF isoforms share a high degree of sequence similarity, they are obviously under different regulation and may have individual functions, mediated by isoform-specific proteinprotein interactions (1,7,8).…”

Unique N-region Determines Low Basal Activity and Limited Inducibility of A-RAF Kinase

“…These differences were much more pronounced in the experiments where full-length RAF kinases were used in binding assays. Furthermore, it was demonstrated that the higher accessibility of Ras for B-RAF is caused by its prolonged N-terminal tail, probably keeping RBD of B-RAF in an "open" conformation, because truncation of this region resulted in a protein that changed its kinase properties and closely resembles C-RAF (47). Taken together, the length of the extreme N termini of RAF kinases plays an important role with regard to RAF activation.…”