Raf-1 Cysteine-Rich Domain Increases the Affinity of K-Ras/Raf at the Membrane, Promoting MAPK Signaling.
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ABSTRACT:
SUBMITTER: Li S
PROVIDER: S-EPMC8183739 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.
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Project description:BackgroundHepatocellular carcinoma (HCC) is one of the most common clinical malignancies quite susceptible to recurrence and metastasis. Despite several improvements in therapeutic approaches, the prognosis remains poor due to the limited treatment options. A bioinformatics analysis based on TCGA databases revealed that the recombinant human L antigen family member 3 (LAGE3) might function as an effective prognostic and diagnostic biomarker for HCC, as LAGE3, a protein-coding gene, maintains several important biological functions and has a physiological significance in the CTAG family while simultaneously being involved in regulating the occurrence and invasion of numerous types of tumors. However, the LAGE3 gene's functional and regulatory mechanism in the progression of HCC remains unclear.MethodsThe LAGE3 level was investigated in 79 HCC tissues cases, ten HCC adjacent tissue cases, and six cases of normal liver tissues by IHC, while the LAGE3 level was evaluated in BEL-7404, SMCC-7721, Huh-7, HepG2, and MIHA cell lines by qRT-PCR and Western blot tests. Although the proliferation, migration, invasion, and apoptotic abilities of HCC cells were measured in vitro after silencing assay to probe the role of LAGE3 in HCC cells, the tumor xenograft growth experiment was used to verify the in vivo effect of LAGE3 gene knockdown on the growth of HCC tumors combined with bioinformatics analysis to study the LAGE3 mechanisms regulating HCC proliferation.ResultsOur results implied that LAGE3 was extensively expressed in HCC cell lines like BEL-7404, SMCC-7721, and Huh-7 cells as well as HCC tissues, but a lower expression was observed in HepG2 cells. Additionally, LAGE3 restrains cellular proliferation, promotes apoptotic pathways in HCC cells, and inhibits the growth of HCC tumors in vivo. Lastly, it was stated that LAGE3 might promote tumor development in HCC via PI3K/AKT/mTOR and Ras/RAF/MAPK pathways.ConclusionThis study shows that the development of specific LAGE3 target drugs might become new effective treatment modalities for HCC patients.
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Project description:During the activation of mitogen-activated protein kinase (MAPK) signaling, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3-5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane that correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.
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