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Proc Natl Acad Sci U S A. 2008 Feb 5;105(5):1442-7. doi: 10.1073/pnas.0708966105. Epub 2008 Jan 30.

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates.

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1
Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA.

Abstract

We describe a method for rapid identification of protein kinase substrates. Cdk1 was engineered to accept an ATP analog that allows it to uniquely label its substrates with a bio-orthogonal phosphate analog tag. A highly specific, covalent capture-and-release methodology was developed for rapid purification of tagged peptides derived from labeled substrate proteins. Application of this approach to the discovery of Cdk1-cyclin B substrates yielded identification of >70 substrates and phosphorylation sites. Many of these sites are known to be phosphorylated in vivo, but most of the proteins have not been characterized as Cdk1-cyclin B substrates. This approach has the potential to expand our understanding of kinase-substrate connections in signaling networks.

PMID:
18234856
PMCID:
PMC2234163
DOI:
10.1073/pnas.0708966105
[Indexed for MEDLINE]
Free PMC Article
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