Chemical Genetic Approach for Kinase‐Substrate Mapping by Covalent Capture of Thiophosphopeptides and Analysis by Mass Spectrometry

Abstract

Mapping kinase‐substrate interactions demands robust methods to rapidly and unequivocally identify substrates from complex protein mixtures. Toward this goal, we present a method in which a kinase, engineered to utilize synthetic ATPγS analogs, specifically thiophosphorylates its substrates in a complex lysate. The thiophosphate label provides a bio‐orthogonal tag that can be used to affinity purify and identify labeled proteins. Following the labeling reaction, proteins are digested with trypsin; thiol‐containing peptides are then covalently captured and non‐thiol‐containing peptides are washed from the resin. Oxidation‐promoted hydrolysis, at sites of thiophosphorylation, releases phosphopeptides for analysis by tandem mass spectrometry. By incorporating two specificity gates—kinase engineering and peptide affinity purification—this method yields high‐confidence substrate identifications. This method gives both the identity of the substrates and phosphorylation‐site localization. With this information, investigators can analyze the biological significance of the phosphorylation mark immediately following confirmation of the kinase‐substrate relationship. Here, we provide an optimized version of this technique to further enable widespread utilization of this technology. Curr. Protoc. Chem Biol. 2:15‐36. © 2010 by John Wiley & Sons, Inc.

Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen

“…As a positive control, we sought to confirm that our WT-TAK1 f would strongly phosphorylate a known substrate, MKK6. MKK6 was not identified by MS because of the presence of cysteine in tryptic MKK6 peptides containing TAK1 phosphorylation sites, as Cys-containing peptides are permanently retained on the capture resin (9). TAK1 strongly phosphorylated kinase-dead MKK6 as shown by the incorporation of 32 P in the WT-TAK1 f -labeled sample ( Fig.…”

“…We first sought to identify direct substrates of TAK1 using a chemical genetic, analog-specific (AS) kinase approach ( Fig. S1A) (8,9). Mutation of a single bulky residue within the active site of a kinase, termed the active-site gatekeeper residue, to alanine or glycine expands the native ATP binding pocket.…”

“…Other studies have suggested a role for TAK1 in directly regulating protein degradation to prevent accumulation of reactive oxygen species (7). Thus, we turned to the analog-specific kinase covalent capture methodology (8,9) to identify direct TAK1 substrates in vitro. Through this method, we identified hundreds of candidate substrates.…”

The G1 Cyclin-dependent Kinase CRK1 in Trypanosoma brucei Regulates Anterograde Protein Transport by Phosphorylating the COPII Subunit Sec31

“…Purification of thiophosphopeptides for mass spectrometric analysis was carried out essentially as described (30,31). The total trypanosome proteins that had been incubated with N 6 -(benzyl)ATP␥S in the presence (experimental sample) or absence (control sample) of the CRK1as-CYC2 complex were digested to peptides with trypsin, and the products were incubated with iodoacetyl-agarose, which allows the thio-containing groups to react to form covalent bonds.…”

Generation and characterization of an analog-sensitive PERK allele

“…38 Following thiophosphorylation, reactions were alkylated by adding p-nitrobenzyl mesylate (Abcam ab138910) to 2.5 mM for 1 h at room temperature. Samples were boiled in SDS sample buffer, run on an SDS-PAGE gel, and probed by western blot for thiophosphorylated protein.…”