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PKC epsilon is associated with myosin IIA and actin in fibroblasts
Karen England1, David Ashford, Daniel Kidd
1Department of Biology, University of York, York YO10 5DD, UK. k.england@ucc.ie
Abstract:
Proteins coimmunoprecipitating with protein kinase C (PKC) epsilon in fibroblasts were identified through matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF m/s). This method identified myosin IIA in PKC epsilon immunoprecipitates, as well as known PKC epsilon binding proteins, actin, beta'Cop and cytokeratin. Myosin is not a substrate for PKC epsilon. Immunofluorescence analysis showed that PKC epsilon is colocalised with actin and myosin in actomyosin stress fibers in fibroblasts. Inhibitors of PKC and myosin ATPase activity, as well as microfilament-disrupting drugs, all inhibited spreading of fibroblasts after passage, suggesting a role for a PKC epsilon-actin-myosin complex in cell spreading.
Insights
Protein kinase C (PKC) epsilon interacts with myosin IIA and actin in fibroblast stress fibers. This PKC epsilon-actin-myosin complex is crucial for cell spreading after passage.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Protein kinase C (PKC) epsilon is involved in various cellular processes.
- The molecular interactions of PKC epsilon in fibroblasts are not fully understood.
Purpose of the Study:
- To identify proteins that coimmunoprecipitate with PKC epsilon in fibroblasts.
- To investigate the role of PKC epsilon in fibroblast cell spreading.
Main Methods:
- Matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) was used to identify coimmunoprecipitating proteins.
- Immunofluorescence microscopy was employed to visualize the localization of PKC epsilon, actin, and myosin.
- Pharmacological inhibitors were used to assess the functional role of PKC and myosin in cell spreading.
Main Results:
- Myosin IIA was identified as a novel binding partner of PKC epsilon, alongside known interactors actin, beta'Cop, and cytokeratin.
- PKC epsilon was found to colocalize with actin and myosin in actomyosin stress fibers within fibroblasts.
- Inhibition of PKC, myosin ATPase activity, or microfilaments impaired fibroblast spreading.
Conclusions:
- PKC epsilon forms a complex with actin and myosin IIA within actomyosin stress fibers.
- This PKC epsilon-actin-myosin complex plays a significant role in fibroblast cell spreading.