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Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay
Published on: January 16, 2017
Gli protein nuclear localization signal.
1Laboratory for Behavioral and Developmental Disorders, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan.
Vitamins and Hormones
|March 7, 2012
Summary
Hedgehog signaling regulates the nuclear transport of Drosophila cubitus interruptus (Ci) and Gli proteins. Importin α3 mediates Ci nuclear import, with its interaction affected by protein cleavage, phosphorylation, and competition from Roadkill protein.
Area of Science:
- Molecular Biology
- Developmental Biology
- Cell Biology
Background:
- Drosophila cubitus interruptus (Ci) and vertebrate glioblastoma (Gli) proteins are transcription factors in the Gli/Glis/Zic ZF protein superfamily.
- These proteins possess zinc finger (ZF) motifs and are crucial components of the hedgehog (Hh) signaling pathway.
- Nuclear transport, regulated by Hh signaling, is essential for the Hh signal transduction pathway, involving nuclear localization signals (NLS) and nuclear export signals (NES).
Purpose of the Study:
- To elucidate the mechanisms governing the nuclear transport of Ci and Gli proteins.
- To identify the specific nuclear transport proteins involved in Ci import.
- To understand how Hh signaling regulates the activity of NLS and NES in Ci and Gli proteins.
Main Methods:
- Mapping of the NLS within the fifth ZF domain and C-terminal side of Gli/Ci proteins.
- Identification of Importin α3 as a nuclear transport protein for Ci.
- 3D structural modeling of the Gli NLS-Importin α complex.
- Investigation of Hh signaling-dependent mechanisms including protein cleavage and phosphorylation.
- Analysis of the role of protein kinase A and the Roadkill protein in regulating Ci nuclear import.
Main Results:
- The NLS of Gli/Ci proteins contains two basic residue clusters, conserved but with variations compared to Glis and Zic proteins.
- Importin α3 binds to the Gli NLS, with structural modeling predicting interaction of basic clusters with Importin α binding interfaces.
- Hh signaling regulates NLS/NES function through protein cleavage (Ci, Gli3) and phosphorylation (Gli1) via protein kinase A.
- The Roadkill protein competes with Ci for Importin α3 binding, inhibiting Ci nuclear import.
Conclusions:
- Nuclear import of Ci and Gli proteins is tightly regulated by specific interactions with Importin α3.
- Hedgehog signaling modulates Ci/Gli nuclear transport through post-translational modifications and protein-protein interactions.
- Understanding these transport mechanisms provides insights into Hh pathway regulation and potential therapeutic targets.
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