Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates.
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.
Blethrow
Justin D
JD
Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA.
Glavy
Joseph S
JS
Morgan
David O
DO
Shokat
Kevan M
KM
D016203:CDC2 Protein Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D016384:Consensus Sequence; D019926:Cyclin B / Q000737:chemistry* / Q000378:metabolism; D056744:Cyclin B1; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010710:Phosphates / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D013379:Substrate Specificity
C534699:CCNB1 protein, human; D002457:Cell Extracts; D019926:Cyclin B; D056744:Cyclin B1; D010710:Phosphates; D010748:Phosphopeptides; D016203:CDC2 Protein Kinase; C035638:thiophosphoric acid