Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.


Sci. Signal., 12 February 2008
Vol. 1, Issue 6, p. ec56
[DOI: 10.1126/stke.16ec56]


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-{gamma}-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]

Citation: N. R. Gough, Capturing Kinase Substrates. Sci. Signal. 1, ec56 (2008).

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882