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Updated: Jul 4, 2025

Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells
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Far-reaching effects of tyrosine64 phosphorylation on Ras revealed with BeF3- complexes

Patrick Baumann1,2,3, Yi Jin4,5,6

  • 1School of Chemistry, Cardiff University, Park Place, Cardiff, CF10 3AT, UK.

Communications Chemistry
|January 31, 2024
PubMed

Insights

Tyrosine phosphorylation of Ras protein by Src kinase alters its conformation, affecting its interactions. This study reveals how phosphorylation at tyrosine64 impacts Ras protein structure and function.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Src kinase-mediated tyrosine phosphorylation of Ras protein is known to disrupt its regulatory and communication pathways.
  • The precise mechanisms by which this phosphorylation influences Ras interactions remain unclear.

Purpose of the Study:

  • To elucidate the structural and mechanistic basis of Ras regulation by tyrosine phosphorylation.
  • To investigate the conformational changes induced by phosphorylation at tyrosine64 of Ras protein.

Main Methods:

  • Quantification of tyrosine64 mono-phosphorylation using 31P Nuclear Magnetic Resonance (NMR) and mutagenesis.
  • Determination of crystal structures of unphosphorylated and phosphorylated Ras-BeF3- complexes.
  • Analysis of conformational changes using 19F NMR.

Main Results:

  • Crystal structures revealed distinct "closed" conformations for phosphorylated Ras, differing from previously observed "open" conformations.
  • Phosphorylation at tyrosine64 induced distant conformational changes, reducing affinity for the downstream effector Raf.
  • 19F NMR indicated altered intrinsic GTPase and nucleotide exchange rates, with both major "closed" and minor "open" conformations present.

Conclusions:

  • Tyrosine64 phosphorylation induces significant conformational changes in Ras protein, impacting its functional interactions.
  • Metal fluoride complexes are effective tools for studying conformational dynamics in modified Ras proteins.
  • The study provides novel mechanistic insights into Ras intrinsic GTP hydrolysis and regulation.

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