Methodology Capturing Kinase Substrates

Nancy R. Gough

Science Signaling, AAAS, Washington, DC 20005, USA

Combining chemistry and molecular biology allowed the development of kinases that use an ATP analog to phosphorylate their targets (so-called as-kinases, for kinases with an analog-sensitizing mutation). However, application of this technique to identify kinase substrates in systems where the substrates are low-abundance proteins has proven challenging. Blethrow et al. describe a selective chemistry-based purification method for identifying low-abundance substrates. The purification process involves phosphorylation of the substrates in cell lysates by an as-kinase that can use the radiolabeled ATP analog [N⁶-(benzyl)ATP--³⁵S] to generate thiophosphopeptides, hydrolysis of the cell lysates to generate peptide fragments, and then two-step purification of the thiophosphopeptides. In the first step, the peptide fragments are mixed with iodoacetyl-agarose resin, which binds to the thiophosphorylated peptides and peptides containing cysteine. In the second step, the thiophosphorylated peptides are selectively released from the resin by oxidation and are then identified by mass spectrometry. The method was applied to HeLa cell lysates phosphorylated by as-cyclin-dependent kinase 1/cyclin B (as-Cdk1-cyclin B). Many known substrates as well as several new substrates and new phosphorylation sites were identified. Analysis of the sequences surrounding the phosphorylation sites revealed that ~30% of the sites did not match the consensus sequence, suggesting that sequence analysis underestimates the number of targets. The method was also applied to a subcellular fraction, the nuclear envelope of rat liver nuclei, and known and new candidate targets that may be regulated by phosphorylation during mitosis. The main limitation of the method is that substrates with cysteine residues will be excluded from the purification.

J. D. Blethrow, J. S. Glavy, D. O. Morgan, K. M. Shokat, Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates. Proc. Natl. Acad. Sci. U.S.A. 105, 1442-1447 (2008). [Abstract] [Full Text]

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PERSPECTIVES
STKE Perspectives
Hitting the Target: Emerging Technologies in the Search for Kinase Substrates

Brendan D. Manning and Lewis C. Cantley (10 December 2002)
Sci. STKE 2002 (162), pe49. [DOI: 10.1126/scisignal.1622002pe49]
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