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Updated: Aug 11, 2025

Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells
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Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells

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Two Closed Conformations of CRAF Require the 14-3-3 Binding Motifs and Cysteine-Rich Domain to be Intact in Live

Kenji Okamoto1, Yasushi Sako1

  • 1Cellular Informatics Laboratory, Cluster for Pioneering Research, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan.

Journal of Molecular Biology
|February 3, 2023
PubMed

Insights

This study reveals how the RAF protein changes shape in live cells, showing that a 14-3-3 dimer binding induces closed, inactive states crucial for cell signaling regulation.

Area of Science:

  • Cellular signaling pathways
  • Molecular dynamics and protein conformation
  • Biophysics of kinase regulation

Background:

  • The Rapidly Accelerated Fibrosarcoma (RAF) kinase is a key component of the RAS-MAPK signaling pathway, regulating cell growth and survival.
  • RAF protein activity is modulated by its conformational states, including open/close dynamics and dimerization, but these remain poorly understood in live cells.
  • Understanding RAF conformation is critical for deciphering its role in signal transduction and developing targeted therapies.

Purpose of the Study:

  • To investigate the structural dynamics and conformational states of cytosolic CRAF in live HeLa cells using single-molecule Förster resonance energy transfer (smFRET).
  • To elucidate the role of 14-3-3 binding in regulating CRAF conformation and activity.
  • To compare the conformational behavior of wild-type (WT)-CRAF with mutant forms and assess responses to epidermal growth factor (EGF) stimulation.

Main Methods:

  • Utilized alternating laser excitation smFRET to measure cytosolic CRAF dynamics in live HeLa cells.
  • Analyzed smFRET distributions of structural states for full-length CRAF, including WT, mutants (14-3-3 binding sites, cysteine-rich domain), and N-terminus truncation.
  • Employed global fitting with three beta distributions to analyze smFRET data and characterize distinct CRAF conformations.

Main Results:

  • Identified distinct smFRET distributions corresponding to different structural states of CRAF in live cells.
  • Demonstrated that a 14-3-3 dimer binds to two sites on a single CRAF molecule, inducing autoinhibitory closed conformations.
  • Observed two distinct closed conformations adopted by the majority of WT-CRAF, exhibiting differential responsiveness to EGF stimulation.

Conclusions:

  • The binding of a 14-3-3 dimer is a critical regulator of CRAF conformation, promoting autoinhibitory closed states.
  • CRAF exists in at least two distinct closed conformations, suggesting a nuanced regulatory mechanism.
  • These findings provide novel insights into CRAF conformational dynamics in response to signaling cues within a cellular context.
Keywords:
ALEXintramolecular complexphosphorylationprotein conformationsingle-molecule FRET

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