Combining chemical genetics and proteomics to identify protein kinase substrates Academic Article uri icon

Overview

MeSH Major

  • Cyclin-Dependent Kinases
  • Genetic Techniques
  • Proteomics
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins

abstract

  • Phosphorylation is a ubiquitous protein modification important for regulating nearly every aspect of cellular biology. Protein kinases are highly conserved and constitute one of the largest gene families. Identifying the substrates of a kinase is essential for understanding its cellular role, but doing so remains a difficult task. We have developed a high-throughput method to identify substrates of yeast protein kinases that employs a collection of yeast strains each expressing a single epitope-tagged protein and a chemical genetic strategy that permits kinase reactions to be performed in native, whole-cell extracts. Using this method, we screened 4,250 strains expressing epitope-tagged proteins and identified 24 candidate substrates of the Pho85-Pcl1 cyclin-dependent kinase, including the known substrate Rvs167. The power of this method to identify true kinase substrates is strongly supported by functional overlap and colocalization of candidate substrates and the kinase, as well as by the specificity of Pho85-Pcl1 for some of the substrates compared with another Pho85-cyclin kinase complex. This method is readily adaptable to other yeast kinases.

publication date

  • December 13, 2005

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC1306798

Digital Object Identifier (DOI)

  • 10.1073/pnas.0509080102

PubMed ID

  • 16330754

Additional Document Info

start page

  • 17940

end page

  • 5

volume

  • 102

number

  • 50