title: In vivo conditions to identify Prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish.
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.
authors: Max Delbrück Center for Molecular Medicine, Berlin, Germany.
authors: Elena
authors: E
authors: Cibrián Uhalte
authors: Marieluise
authors: M
authors: Kirchner
authors: Nicole
authors: N
authors: Hellwig
authors: Jasmina J
authors: JJ
authors: Allen
authors: Stefan
authors: S
authors: Donat
authors: Kevan M
authors: KM
authors: Shokat
authors: Matthias
authors: M
authors: Selbach
authors: Salim
authors: S
authors: Abdelilah-Seyfried
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D004625:Embryo, Nonmammalian / Q000201:enzymology; D057075:Enzyme Assays / Q000379:methods*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism*; D000099293:Protein Kinase C-lambda
chemical_list: D000255:Adenosine Triphosphate; D007527:Isoenzymes; D050505:Mutant Proteins; D011493:Protein Kinase C; D013438:Sulfhydryl Compounds; D000099293:Protein Kinase C-lambda; C022571:adenosine 5'-O-(3-thiotriphosphate)