Related Experiment Videos
Chromosomal passenger complex hydrodynamics suggests chaperoning of the inactive state by nucleoplasmin/nucleophosmin
Mariah L Hanley1,2, Tae Yeon Yoo3, Matthew Sonnett1
1Department of Systems Biology, Harvard Medical School, Boston, MA 02114-5701.
Abstract:
The chromosomal passenger complex (CPC) is a conserved, essential regulator of cell division. As such, significant anti-cancer drug development efforts have been focused on targeting it, most notably by inhibiting its AURKB kinase subunit. The CPC is activated by AURKB-catalyzed autophosphorylation on multiple subunits, but how this regulates CPC interactions with other mitotic proteins remains unclear. We investigated the hydrodynamic behavior of the CPC in Xenopus laevis egg cytosol using sucrose gradient sedimentation and in HeLa cells using fluorescence correlation spectroscopy. We found that autophosphorylation of the CPC decreases its sedimentation coefficient in egg cytosol and increases its diffusion coefficient in live cells, indicating a decrease in mass. Using immunoprecipitation coupled with mass spectrometry and immunoblots, we discovered that inactive, unphosphorylated CPC interacts with nucleophosmin/nucleoplasmin proteins, which are known to oligomerize into pentamers and decamers. Autophosphorylation of the CPC causes it to dissociate from nucleophosmin/nucleoplasmin. We propose that nucleophosmin/nucleoplasmin complexes serve as chaperones that negatively regulate the CPC and/or stabilize its inactive form, preventing CPC autophosphorylation and recruitment to chromatin and microtubules in mitosis.
Insights
The chromosomal passenger complex (CPC) undergoes autophosphorylation, reducing its mass and releasing it from nucleophosmin/nucleoplasmin chaperones. This process regulates CPC activity during cell division and has implications for anti-cancer drug development.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The chromosomal passenger complex (CPC) is crucial for cell division regulation.
- Targeting the CPC, particularly its AURKB kinase subunit, is a focus of anti-cancer drug development.
- Mechanisms regulating CPC interactions with other proteins during mitosis are not fully understood.
Purpose of the Study:
- To investigate how CPC autophosphorylation affects its mass and interactions with other proteins.
- To elucidate the role of nucleophosmin/nucleoplasmin in CPC regulation.
Main Methods:
- Sucrose gradient sedimentation in Xenopus laevis egg cytosol.
- Fluorescence correlation spectroscopy in live HeLa cells.
- Immunoprecipitation coupled with mass spectrometry and immunoblots.
Main Results:
- CPC autophosphorylation decreases its sedimentation coefficient and increases its diffusion coefficient, indicating reduced mass.
- Inactive CPC binds to nucleophosmin/nucleoplasmin proteins.
- Active, phosphorylated CPC dissociates from nucleophosmin/nucleoplasmin.
Conclusions:
- Nucleophosmin/nucleoplasmin complexes act as chaperones, negatively regulating the CPC.
- These chaperones stabilize the inactive form of the CPC, preventing premature activation and localization during mitosis.
- Understanding this regulatory mechanism offers new insights for anti-cancer therapies targeting cell division.