Search for Kinases

Kinases and their substrates are central components of signaling pathways. Most of the time, it is either known that a specific kinase is involved in a certain signaling pathway, or it is known that a certain protein of interest is being phosphorylated under a given condition. Thus, either the substrates of the kinase or the kinase itself remains to be identified. Candidates for kinase substrates have in the past been successfully identified by genetic screens and epistasis analyses or laborious biochemical purifications and in vitro assays (Manning and Cantley 2002).

Recently, an elegant method has been developed, which makes use of a series of synthetic peptides comprising the phosphorylation site of a protein of interest and the detection of changes in phosphorylation status of these standard peptides by mass spectrometry. In this approach, the standard peptides are incubated with fractions of cell extract and the phosphorylation status of the standard peptides is analyzed by multiple reaction monitoring in a triple-quadrupole mass spectrometer (Glinski et al. 2003; Glinski and Weckwerth 2005). This strategy was applied in a study to identify kinases involved in phosphorylation of trehalose-6-phosphate synthase (Glinski and Weckwerth 2005), and in addition it allowed testing for various parameters required for the kinase activity, such as calcium dependence. By carrying out the kinase

Fig. 3 Principle of kinase assay. Protein extract from plants is fractionated, e.g., by size exclusion chromatography. Each fraction is incubated with one or multiple target peptides of interest. The target peptides are chosen around potential phosphorylation sites and they are used in the assay in their unphosphorylated form. After incubation of protein extract fractions with the target peptide, phosphorylation of the target peptide is analyzed by mass spectrometry. The full analysis of proteins present in the fraction showing kinase activity will identify potential candidates for that kinase activity

Fig. 3 Principle of kinase assay. Protein extract from plants is fractionated, e.g., by size exclusion chromatography. Each fraction is incubated with one or multiple target peptides of interest. The target peptides are chosen around potential phosphorylation sites and they are used in the assay in their unphosphorylated form. After incubation of protein extract fractions with the target peptide, phosphorylation of the target peptide is analyzed by mass spectrometry. The full analysis of proteins present in the fraction showing kinase activity will identify potential candidates for that kinase activity assays on fractionated cell extract, the mass spectrometric analysis of the respective fractions exhibiting kinase activity can be used to narrow down the protein candidates for this kinase activity (Fig. 3).

The reverse approach, in which the kinase is known and the substrate is unknown, can be tackled by the use of peptide arrays. Synthetic pep-tides comprising a set of known and predicted phosphorylation sites from a variety of proteins are spotted on nylon membranes or glass slides. Upon incubation with the purified kinase of interest and the incorporation of radioactive or fluorescent phosphate analogs, the substrate specificity of the kinase can be determined. This approach is especially suitable for identifying the motif specificity of several kinases (Houseman et al. 2002). In plants, this method will gain more importance in the future, as the library of experimental phosphorylation sites is growing continuously, and may provide a basis for targeted array analysis.

An elegant method of finding specific kinase-substrate pairs in vivo involves genetic manipulation of the kinase pocket and the use of the respective synthetic ATP analogs (Shah et al. 1997). In that way, the genetically manipulated kinase can only use the synthetic ATP analog in its reactions, and the substrate proteins modified with the ATP analog can be affinity purified and identified by mass spectrometry. This strategy has been applied to a large-scale screen for specific substrates to the Pho85-Pcl1 cyclin-dependent kinase in yeast (Dephoure et al. 2005). Given the vast collections of knockout mutants, such a screen would certainly also be feasible in Arabidopsis.

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