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PNAS 105 (5): 1442-1447

Copyright © 2008 by the National Academy of Sciences.


PHYSICAL SCIENCES / BIOLOGICAL SCIENCES / CHEMISTRY / BIOCHEMISTRY

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

Justin D. Blethrow*,{dagger}, Joseph S. Glavy{ddagger},§, David O. Morgan, and Kevan M. Shokat*,||

*Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158; {ddagger}Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, New York, NY 10065; and Departments of Physiology and Biochemistry and Biophysics, University of California, San Francisco, CA 94158

Edited by Benjamin Cravatt, The Scripps Research Institute, La Jolla, CA, and accepted by the Editorial Board November 29, 2007

Received for publication September 21, 2007.

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.

Key Words: chemical biology • chemical genetics • cyclin-dependent • phosphorylation • signaling


Author contributions: J.D.B., D.O.M., and K.M.S. designed research; J.D.B. performed research; J.D.B. contributed new reagents/analytic tools; J.D.B. and J.S.G. analyzed data; and J.D.B. and K.M.S. wrote the paper.

{dagger}Present address: ThermoFisher Scientific, San Jose, CA 95134.

§Present address: Stevens Institute of Technology, Department of Chemistry and Chemical Biology, Hoboken, NJ 07030.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. B.C. is a guest editor invited by the Editorial Board.

Data Deposition: Raw MS data can be accessed at ftp://acbishop.ucsf.edu

This article contains supporting information online at www.pnas.org/cgi/content/full/0708966105/DC1.

||To whom correspondence should be addressed. E-mail: shokat{at}cmp.ucsf.edu

© 2008 by The National Academy of Sciences of the USA


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