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Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
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A complex of Shc and Ran-GTPase localises to the cell nucleus

R George1, H-L Chan, Z Ahmed

  • 1Department of Biochemistry and Molecular Biology, University College London, UK.

Cellular and Molecular Life Sciences : CMLS
|January 21, 2009
PubMed

Insights

Researchers identified the nuclear transport protein Ran-GTPase as a binding partner for p52 Shc. This interaction, along with the nuclear transport factor NTF2, suggests a novel mechanism for Shc protein nuclear import.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The adaptor protein Shc exists in three isoforms with varied roles in cell signaling.
  • The p46 Shc isoform has been observed in the nuclei of hepatocellular carcinoma cells, indicating a non-cytoplasmic function.
  • Mechanisms and ligands facilitating Shc isoform nuclear transport remain largely uncharacterized.

Purpose of the Study:

  • To identify ligands responsible for the nuclear transport of Shc isoforms.
  • To elucidate the molecular interactions governing Shc nuclear localization.

Main Methods:

  • Far western blotting was employed to determine the p52 Shc interactome.
  • In vitro binding assays assessed the interaction between p52 Shc and identified proteins.
  • Co-immunoprecipitation, pull-down assays, and fluorescence lifetime imaging microscopy (FLIM) were used in stable cell lines to confirm interactions and localization.

Main Results:

  • The Ran-GTPase nuclear transport protein was identified as a binding partner for p52 Shc, exhibiting low micromolar affinity in vitro.
  • Cellular interaction and nuclear localization of Shc were confirmed through co-immunoprecipitation, pull-down, and FLIM experiments.
  • The nuclear transport factor NTF2 was found to form a complex with both Ran and Shc.

Conclusions:

  • Ran-GTPase directly interacts with p52 Shc.
  • The formation of a tertiary complex involving Ran, NTF2, and Shc suggests a novel pathway for Shc nuclear import.
  • This finding provides insight into the distinct roles of Shc isoforms in cellular processes, particularly in the context of cancer cells.

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