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Updated: May 20, 2026
Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos
Published on: February 19, 2016
In vivo conditions to identify Prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish
Elena Cibrián Uhalte1, Marieluise Kirchner, Nicole Hellwig
1Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Abstract:
Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins in their cellular context. The analog-sensitive kinase method provides a powerful tool for the identification of kinase substrates under in vivo conditions. However, it has remained a major challenge to establish screens based on this method in multicellular model organisms. Here, we report the methodology for in vivo conditions using the analog-sensitive kinase method in a genetically-tractable vertebrate model organism, the zebrafish. With this approach, kinase substrates can uniquely be labeled in the developing zebrafish embryo using bulky ATPγS analogs which results in the thiophosphorylation of substrates. The labeling of kinase substrates with a thiophosphoester epitope differs from phosphoesters that are generated by all other kinases and allows for an enrichment of thiophosphopeptides by immunoaffinity purification. This study provides the foundation for using the analog-sensitive kinase method in the context of complex vertebrate development, physiology, or disease.
Insights
Researchers developed a new method to identify protein kinase C iota substrates in zebrafish embryos. This technique uses analog-sensitive kinase technology for in vivo studies, advancing our understanding of kinase functions in development and disease.
Area of Science:
- Cell Biology
- Molecular Biology
- Developmental Biology
Background:
- Protein kinase C iota (PKCι) is crucial for epithelial polarity and organ development.
- Identifying specific kinase substrates is essential for understanding PKCι function.
- Existing methods for identifying kinase substrates are challenging in multicellular organisms.
Purpose of the Study:
- To establish a methodology for identifying kinase substrates in vivo using the analog-sensitive kinase (ASK) method in zebrafish.
- To enable mechanistic studies of PKCι in a complex vertebrate model.
Main Methods:
- Utilized analog-sensitive kinase technology in zebrafish embryos.
- Employed bulky ATPγS analogs for unique thiophosphorylation of PKCι substrates.
- Developed immunoaffinity purification for thiophosphopeptides.
Main Results:
- Successfully labeled and enriched PKCι kinase substrates in developing zebrafish embryos.
- Demonstrated the feasibility of the ASK method in a vertebrate model organism.
- Established a method to distinguish PKCι substrates from those of other kinases.
Conclusions:
- The developed methodology provides a foundation for studying kinase substrates in vivo in zebrafish.
- This approach facilitates research into complex vertebrate development, physiology, and disease.
- Enables detailed mechanistic insights into the roles of specific kinases like PKCι.
