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The tumor-sensitive calmodulin-like protein is a specific light chain of human unconventional myosin X
1Tumor Biology Program, Department of Biochemistry and Molecular Biology, Mayo Graduate School and Mayo Clinic Cancer Center, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.
Abstract:
Human calmodulin-like protein (CLP) is an epithelial-specific Ca(2+)-binding protein whose expression is strongly down-regulated in cancers. Like calmodulin, CLP is thought to regulate cellular processes via Ca(2+)-dependent interactions with specific target proteins. Using gel overlays, we identified a approximately 210-kDa protein binding specifically and in a Ca(2+)-dependent manner to CLP, but not to calmodulin. Yeast two-hybrid screening yielded a CLP-interacting clone encoding the three light chain binding IQ motifs of human "unconventional" myosin X. Pull-down experiments showed CLP binding to the IQ domain to be direct and Ca(2+)-dependent. CLP interacted strongly with IQ motif 3 (K(d) approximately 0.5 nm) as determined by surface plasmon resonance. Epitope-tagged myosin X was localized preferentially at the cell periphery in MCF-7 cells, and CLP colocalized with myosin X in these cells. Myosin X was able to coprecipitate CLP and, to a lesser extent, calmodulin from transfected COS-1 cells, indicating that CLP is a specific light chain of myosin X in vivo. Because unconventional myosins participate in cellular processes ranging from membrane trafficking to signaling and cell motility, myosin X is an attractive CLP target. Altered myosin X regulation in (tumor) cells lacking CLP may have as yet unknown consequences for cell growth and differentiation.
Insights
Human calmodulin-like protein (CLP) binds to unconventional myosin X. This interaction, crucial for cell regulation, is calcium-dependent and may be disrupted in cancers lacking CLP.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Human calmodulin-like protein (CLP) is an epithelial-specific calcium-binding protein.
- CLP expression is significantly reduced in various cancers.
- CLP regulates cellular functions through calcium-dependent protein interactions, similar to calmodulin.
Purpose of the Study:
- To identify proteins that interact with human calmodulin-like protein (CLP).
- To investigate the nature and specificity of the interaction between CLP and its binding partners.
- To determine the functional implications of CLP-protein interactions in cellular processes.
Main Methods:
- Gel overlay assays to identify CLP-binding proteins.
- Yeast two-hybrid screening to identify CLP-interacting partners.
- Pull-down assays and surface plasmon resonance to confirm direct binding and determine affinity.
- Immunofluorescence microscopy and co-precipitation assays to assess in vivo interaction and localization.
Main Results:
- A 210-kDa protein was identified that binds CLP specifically and calcium-dependently.
- Yeast two-hybrid screening identified unconventional myosin X's IQ motifs as CLP-binding domains.
- CLP directly binds to the IQ domain of myosin X in a calcium-dependent manner, with high affinity for IQ motif 3.
- CLP colocalizes with myosin X at the cell periphery and functions as a specific light chain for myosin X in vivo.
Conclusions:
- CLP specifically binds to unconventional myosin X, acting as its light chain.
- Myosin X is a likely in vivo target for CLP-mediated regulation.
- Dysregulation of myosin X by CLP loss in cancer may impact cell motility, signaling, and growth.