In Vitro Reconstitution Studies of RAS-Mediated RAF Activation at the Cell Membrane
Bibliographic record
Abstract
Activation of the kinase RAF by the small GTPase RAS at the cell membrane is a key regulatory step in the RAS-MAPK (mitogen-activated protein kinase) signalling pathway, which drives cell growth and is frequently dysregulated in human disease, especially cancer. Efforts to target RAF therapeutically have been complicated by limitations in our understanding of the complexity underlying its activation. RAS and the membrane play an essential role in facilitating the transition of RAF from an auto-inhibited monomer to an active dimer, a process which requires concurrent rearrangement of its regulatory binding partner 14-3-3. However, the mechanistic details underlying these conformational changes remain unclear due to a lack of information on the interaction of full-length, 14-3-3-bound RAF with RAS and the membrane. Here, the membrane-bound signalling complex comprising full-length active dimeric BRAF, 14-3-3 and KRAS4B was reconstituted in vitro for the first time using RAS-conjugated nanodiscs, facilitating the study of critical protein-membrane regulatory interactions. This system was leveraged to examine how membrane lipid composition and spatial density of RAS on the membrane impact the binding affinities of monomeric and dimeric BRAF:14-3-3 complexes for membrane-tethered RAS, and to dissect the effects of RAS and the membrane on the activity of BRAF:14-3-3 complexes. Negative stain and cryogenic electron microscopy imaging was furthermore employed to investigate the structural organization of the active dimeric BRAF:14-3-3:KRAS-nanodisc complex. Through using cell-free, defined lipid bilayers, the findings in this thesis offer a unique biophysical perspective on the activation of RAF by RAS at the membrane.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".