Reversible protein phosphorylation is one of the most common posttranslational modifications and regulates virtually all cellular processes. Protein kinases are among the largest known gene families with more than 500 human kinase genes that comprise nearly 2% of the open reading frames of the human genome [1
]. Moreover, approximately 30% of all cellular proteins are phosphorylated [2
]. The large number of kinases and their substrates make it very difficult to determine which proteins are phosphorylated by specific kinases in vivo
, but this information is critical to understanding kinase functions and the control of biological processes in general. Various strategies have been developed to identify protein kinase substrates, and several have resulted from recent technological advances in substrate detection.
Some approaches have utilized antibodies against phosphomotifs within the substrate proteins as affinity reagents to enrich for phosphorylated peptides. Examples include using antibodies that recognize conserved motifs that are highly specific to a particular kinase [3
], as well as the use of phosphomotif antibodies combined with changes in physiological conditions that stimulate kinase function [4
]. However, it is difficult to apply these approaches to kinases that phosphorylate broader substrate motifs, since there is less epitope conservation among substrates. Another recent method combined quantitative phosphoproteomics with kinase knock-outs and cellular perturbations to identify kinase targets in yeast [5
]. However, with these cell-based approaches, it is often difficult to determine if the putative substrates are direct kinase targets. An in vitro
approach employing arrays of proteins phosphorylated by isolated recombinant kinases has been successfully used in a global analysis of yeast kinase substrates, but this strategy may be difficult to apply to organisms with larger proteomes [6
A 'chemical genetic' approach developed by Kevan Shokat's laboratory addresses many of these potential problems [7
]. In this technique, the kinase to be studied is mutated by replacing a conserved bulky residue within the ATP-binding pocket with a smaller residue. This creates an enlarged ATP binding pocket that enables the mutant kinase to utilize bulky ATP analogues that cannot be used by wild-type cellular kinases, thereby isolating the activity of the mutant kinase from all other cellular kinases [8
]. This technique is broadly applicable to most protein kinases and has led to important advances in substrate identification, including the description of a large number of potential cyclin-dependent kinase (CDK) substrates in yeast [9
]. However, several technical hurdles add substantial challenges when applying this approach to mammalian kinases with broad substrate networks.
In this study, we report a method employing the Shokat strategy to identify direct cyclin A-CDK2 substrates in human cell lysates. CDK2 is activated by both the cyclin E and cyclin A subunits, and cyclin A-CDK2 plays critical roles in cell cycle control, primarily in G1 and S-phases [16
]. We used an engineered cyclin A-CDK2 and ATP-γ-S analogue to label proteins with thiophosphates in cell lysates, and after digestion of the protein mixtures, we employed a single-step chemical enrichment procedure to selectively isolate thiophosphorylated peptides. As these studies were nearing completion, Blethrow et al
] independently reported a similar approach employing engineered CDK1 and thiophosphate enrichment methods that they used to identify a group of 68 putative cyclin B-CDK1 substrates within Hela cell lysates.
We identified 180 proteins and over 220 phosphopeptides that were phosphorylated in cell lysates by cyclin A-CDK2, and these proteins represented diverse cellular pathways. To validate these methods, we selected several candidate substrates and confirmed that they were phosphorylated by cyclin A-CDK2 in vitro on the same sites that we identified in the screen. Finally, we selected one novel substrate, the ribosomal protein RL12, for further study: site-directed mutagenesis and phosphopeptide mapping confirmed that CDK2 phosphorylates RL12 in vitro and in vivo on the same site determined by our methods.