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CARMIL is a bona fide capping protein interactant.
Kirsten Remmert1, Thomas E Olszewski, M Blair Bowers
1Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
The Journal of Biological Chemistry
|November 5, 2003
Summary
CARMIL protein interacts with capping protein and self-associates. These interactions are significant at cellular concentrations, suggesting physiological relevance for CARMIL function in Acanthamoeba.
Area of Science:
- Cell Biology
- Biochemistry
- Protein Interactions
Background:
- CARMIL (Acan 125) is a multidomain protein identified by its interaction with myosin SH3 domains.
- Previous studies suggested CARMIL binds capping protein and the Arp2/3 complex.
Purpose of the Study:
- To biochemically characterize the interaction between Acanthamoeba CARMIL and capping protein.
- To determine the binding site and affinity of CARMIL for capping protein.
- To investigate the self-association properties of CARMIL.
Main Methods:
- Copurification and microsequencing to identify interacting partners.
- Chemical cross-linking, size exclusion chromatography, and analytical ultracentrifugation.
- Surface plasmon resonance and SH3 domain pull-down assays.
- Rotary shadowed electron microscopy.
Main Results:
- Acanthamoeba CARMIL tightly interacts with capping protein subunits (alpha and beta).
- The dissociation constant for CARMIL-capping protein interaction is approximately 0.4 µM or lower.
- CARMIL's proline-rich domain binds capping protein.
- CARMIL exists in a monomer-dimer equilibrium with an association constant of 1.0 x 10^6 M^-1.
Conclusions:
- CARMIL self-associates and binds capping protein with high affinity.
- These interactions are likely physiologically relevant given cellular protein concentrations.
- CARMIL's dual interaction capabilities suggest a role in regulating cellular processes.