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Molecular cloning and functional characterization of mouse coactosin-like protein
Johanne Doucet1, Patrick Provost, Bengt Samuelsson
1Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, S-171 77, Sweden.
Abstract:
Coactosin was first isolated from Dictyostelium discoideum and, as reported, human coactosin-like protein (CLP) was identified in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. A mouse CLP (mCLP) cDNA clone was identified among EMBL/GenBank EST sequences. The derived amino acid sequence (142 residues) was 95.1% identical with human CLP. Here, we also show that mCLP interacts with actin and 5LO in the two-hybrid system. High-speed cosedimentation assays and GST-binding assays confirmed these protein interactions. In chemical cross-linking experiments, one molecule of mCLP was covalently linked to either one subunit of actin or one molecule of 5LO. The mCLP-F-actin and mCLP-5LO associations were pH-insensitive and Ca(2+)-independent. However, association with actin was best observed at low salt concentrations, while association with 5LO was favored by salt, indicating different binding characteristics.
Insights
Mouse coactosin-like protein (mCLP) interacts with actin and 5-lipoxygenase (5LO). These protein interactions were confirmed through various assays, revealing distinct binding characteristics based on salt concentration.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Coactosin, initially isolated from Dictyostelium discoideum, has homologs in higher eukaryotes.
- Human coactosin-like protein (CLP) was previously identified through a yeast two-hybrid screen with 5-lipoxygenase (5LO).
Purpose of the Study:
- To identify and characterize mouse coactosin-like protein (mCLP).
- To investigate the interactions of mCLP with actin and 5LO.
Main Methods:
- Yeast two-hybrid system to screen for protein interactions.
- High-speed cosedimentation and GST-binding assays to confirm interactions.
- Chemical cross-linking experiments to determine stoichiometry of binding.
Main Results:
- Mouse CLP (mCLP) shares 95.1% amino acid identity with human CLP.
- mCLP was shown to interact with both actin and 5LO.
- Interactions were pH-insensitive and Ca(2+)-independent, with differing salt concentration preferences for actin versus 5LO binding.
Conclusions:
- mCLP interacts with both actin and 5LO, suggesting a role in cellular processes involving these proteins.
- The differential salt dependence indicates distinct binding mechanisms for actin and 5LO.
- Further research is warranted to elucidate the functional significance of mCLP in mammalian cells.