PUBLICATION

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

Authors
Cibrián Uhalte, E., Kirchner, M., Hellwig, N., Allen, J.J., Donat, S., Shokat, K.M., Selbach, M., and Abdelilah-Seyfried, S.
ID
ZDB-PUB-120709-6
Date
2012
Source
PLoS One   7(6): e40000 (Journal)
Registered Authors
Abdelilah-Seyfried, Salim
Keywords
none
MeSH Terms
  • Enzyme Assays/methods*
  • Sulfhydryl Compounds/metabolism
  • Zebrafish/embryology
  • Zebrafish/metabolism*
  • Animals
  • Molecular Sequence Data
  • Embryo, Nonmammalian/enzymology
  • Isoenzymes/chemistry
  • Isoenzymes/metabolism*
  • Substrate Specificity
  • Adenosine Triphosphate/analogs & derivatives
  • Adenosine Triphosphate/metabolism
  • Amino Acid Sequence
  • Mutant Proteins/chemistry
  • Phosphorylation
  • Protein Kinase C/chemistry
  • Protein Kinase C/metabolism*
(all 17)
PubMed
22768194 Full text @ PLoS One
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.
Genes / Markers
Figures
Figure Gallery (3 images)
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Expression
Phenotype
No data available
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
twu34TgTransgenic Insertion
    1 - 1 of 1
    Show
    Human Disease / Model
    No data available
    Sequence Targeting Reagents
    Target Reagent Reagent Type
    prkciMO1-prkciMRPHLNO
    1 - 1 of 1
    Show
    Fish
    Fish
    twu34Tg
    1 - 1 of 1
    Show
    Antibodies
    Orthology
    No data available
    Engineered Foreign Genes
    Marker Marker Type Name
    EGFPEFGEGFP
    1 - 1 of 1
    Show
    Mapping
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    Citation
    Cibrián Uhalte, E., Kirchner, M., Hellwig, N., Allen, J.J., Donat, S., Shokat, K.M., Selbach, M., and Abdelilah-Seyfried, S. (2012) In vivo conditions to identify prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish. PLoS One. 7(6):e40000.