Related Experiment Video

Updated: Dec 25, 2025

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein
07:08

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Published on: January 16, 2020

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Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety

Benjamin M M Grant1,2, Masahiro Enomoto1, Sung-In Back1,2

  • 1Princess Margaret Cancer Center, University Health Network, Toronto, Ontario M5G 1L7, Canada.

Science Signaling
|April 3, 2020
PubMed

Insights

Calcium signaling regulates KRAS4b protein localization. Calmodulin (CaM) binds KRAS4b

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Structural Biology

Background:

  • KRAS4b, a GTPase, controls cell signaling pathways crucial for proliferation, differentiation, and survival.
  • KRAS4b localization to the plasma membrane is essential for its function, involving prenylation and interaction with effector proteins like RAF kinases.
  • The role of calmodulin (CaM) in regulating KRAS4b localization via Ca2+-dependent interactions remains debated.

Purpose of the Study:

  • To elucidate the structural basis and functional consequences of the Ca2+-dependent interaction between CaM and KRAS4b.
  • To investigate the dynamic translocation of KRAS4b in response to Ca2+ signaling in live cells.

Main Methods:

  • Determined the crystal structure of the CaM-KRAS4b complex, supported by solution nuclear magnetic resonance (NMR).
  • Engineered fluorescence resonance energy transfer (FRET)-based biosensor probes (CaMeRAS) for real-time monitoring of KRAS4b translocation.
  • Utilized live HeLa and HEK293 cells to observe Ca2+-dependent KRAS4b dynamics upon stimulation.

Main Results:

  • The crystal structure revealed CaM sequesters KRAS4b's prenyl group in its hydrophobic pocket in a Ca2+-dependent manner.
  • FRET biosensor data demonstrated reversible KRAS4b translocation from the plasma membrane to the cytoplasm upon Ca2+ influx.
  • This Ca2+-CaM-mediated translocation was observed in live cells stimulated by extracellular ligands.

Conclusions:

  • Established a structural and functional mechanism for Ca2+-dependent regulation of KRAS4b localization by CaM.
  • Revealed that Ca2+ signaling can inhibit KRAS4b activity by promoting its cytoplasmic sequestration.
  • Provides insights into how cellular Ca2+ levels modulate KRAS4b's role in signal transduction.

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