Helmholtz Gemeinschaft

Search
Browse
Statistics
Feeds

In vivo conditions to identify prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish

[img] PDF - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
336kB

Item Type:Article
Title:In vivo conditions to identify prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish
Creators Name:Cibrian Uhalte, E. and Kirchner, M. and Hellwig, N. and Allen, J.J. and Donat, S. and Shokat, K.M. and Selbach, M. and Abdelilah-Seyfried, S.
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 ATP{gamma}S 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.
Keywords:Adenosine Triphosphate, Amino Acid Sequence, Enzyme Assays, Isoenzymes, Molecular Sequence Data, Mutant Proteins, Nonmammalian Embryo, Phosphorylation, Protein Kinase C, Substrate Specificity, Sulfhydryl Compounds, Animals, Zebrafish
Source:PLoS ONE
ISSN:1932-6203
Publisher:Public Library of Science (U.S.A.)
Volume:7
Number:6
Page Range:e40000
Date:29 June 2012
Official Publication:https://doi.org/10.1371/journal.pone.0040000
PubMed:View item in PubMed

Repository Staff Only: item control page

Downloads

Downloads per month over past year

Open Access
MDC Library