Raf-1 Cysteine-Rich Domain Increases the Affinity of K-Ras/Raf at the Membrane, Promoting MAPK Signaling

Abstract

K-Ras4B preferentially activates Raf-1. The high-affinity interaction of Ras-binding domain (RBD) of Raf with Ras was solved, but the relative position of Raf's cysteine-rich domain (CRD) in the Ras/Raf complex at the membrane and key question of exactly how it affects Raf signaling are daunting. We show that CRD stably binds anionic membranes inserting a positively charged loop into the amphipathic interface. Importantly, when in complex with Ras/RBD, covalently connected CRD presents the same membrane interaction mechanism, with CRD locating at the space between the RBD and membrane. To date, CRD's role was viewed in terms of stabilizing Raf-membrane interaction. Our observations argue for a key role in reducing Ras/RBD fluctuations at the membrane, thereby increasing Ras/RBD affinity. Even without K-Ras, via CRD, Raf-1 can recruit to the membrane; however, by reducing the Ras/RBD fluctuations and enhancing Ras/RBD affinity at the membrane, CRD promotes Raf's activation and MAPK signaling over other pathways.

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

“…This observation suggests that KRAS-RBD complex is likely to be flexible in the absence of CRD, and this rotation of RBD may be needed to properly orient CRD to stabilize the KRAS-RBDCRD interaction. This is consistent with an MD simulation study which suggested that CRD reduces the fluctuations of the KRAS-RBD complex at the membrane and enhances its binding affinity 24 .…”

Crystal structure reveals the full Ras:Raf interface and advances mechanistic understanding of Raf activation

“…The allosteric connections linking residues D113 at the galectin binding pocket of each Raf-RBD molecule in the dimer, which also includes residue D117 (46) ( Table S2, S3 & S4), leads to a model where Ras/Raf-RBD_CRD dimers couple with galectin dimers to form a higher order macromolecular platform involved in signal amplification and kinetic proofreading ( Interestingly, the location of the Raf-CRD in the context of the Ras/Raf dimer, combined with evidence supporting an approximately perpendicular orientation of helices 3, 4 and 5 with respect to the membrane (38,39), precludes insertion of the Raf-CRD into the membrane. In light of our Ras/Raf-RBD_CRD structure and the increasing evidence that the helix 4/helix 5 Ras dimer is an essential unit for signaling through Ras/Raf/MEK/ERK (34,(38)(39)(40)(41), we must question the presumed membrane-binding function of the Raf-CRD that has been characterized only in the context of monomeric Ras/Raf-RBD_CRD complexes (17,18,20). Instead we suggest that the primary function of previously proposed hydrophobic membrane-binding region, conserved across Raf isoforms, is to stabilize Raf autoinhibition in the absence of Ras as demonstrated in the inactive BRaf/MEK1/14-3-3 cryo-EM structure (27).…”

“…The Raf-CRD requires coordination of two zinc ions and has been shown to interact with various phospholipids and supported lipid bilayers in vitro (3,(13)(14)(15)(16). Its membrane-binding role has further been studied by molecular dynamics (MD) simulations and recently supported by NMR guided characterization of nanodisc-bound KRas/CRaf-RBD_CRD complexes (17)(18)(19)(20)(21)(22).…”

Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells

“…Farnesylated, human influenza hemagglutinin (HA)-tagged K-Ras (G12V), H-Ras (G12V), and N-Ras (G12V) were purified from the membrane-containing fraction of baculovirus-infected Sf 9 insect cells, immobilized on anti-HA para-magnetic beads, and subsequently incubated with PANC-1 cell lysate to precipitate Ras isoform-specific binding proteins. K-Ras4B is referred to as K-Ras since the expression of K-Ras4A is almost undetectable in human lung and pancreatic carcinoma cell lines [33]. Figure 1A shows a representative silver-stained SDS-polyacrylamide gel with the characteristic band pattern for each Ras isoform precipitate.…”