Bio-orthogonal Affinity Purification of Direct Kinase Substrates

Abstract

Protein kinases mediate signal transduction through phosphorylation of their protein substrates. 1 Up to one-third of proteins in a cell are phosphorylated, 2 and a major goal of phosphoproteomics is to characterize phosphorylation mediated signaling cascades by identifying phosphorylated proteins. This feat is analytically challenging because most phosphoproteins are of low abundance and substoichiometrically phosphorylated. Further, once a phosphoprotein is identified, it is difficult to integrate the role of the phosphorylation event into signal transduction networks without knowledge of the upstream kinase. Affinity purification techniques, such as strong cation exchange (SCX), 3 immobilized metal ion affinity chromatography (IMAC), 4 chemical tagging of phosphorylated residues with biotin, 5 and phosphomotif specific antibodies 6 can enrich phosphopeptides or proteins, but the information regarding the kinase responsible for phosphate transfer is uncoupled from the phosphorylation event. We previously have described methodology that allows selective labeling of direct kinase substrates, using analogue specific (as) kinases and orthogonal unnatural nucleotides (A*TP and A*TPγS); 7 however, it remains challenging to biochemically isolate the labeled substrates, impeding their identification.Here, we report a technique that combines direct substrate labeling with immunoaffinity purification (Schemes 1 and 2). To label the substrates of a given kinase, an as allele is used to enzymatically label substrates with A*TPγS. The selectively introduced thiophosphate is then chemically derivatized to construct a bio-orthogonal affinity tag. This approach is similar to other bio-orthogonal tagging strategies using ketones 8 or azides, 9 except thiophosphate cannot be selectively tagged in a single chemical step. For example, an alkylating agent will label both thiophosphate and other cellular nucleophiles, but we envisioned that an antibody could discriminate the thiophosphate alkylation products from other undesired alkylation products. The alkylating agent p-nitrobenzylmesylate (PNBM) was selected to construct the epitope because we predicted antibodies could recognize the product of thiophosphate alkylation over other nitrobenzyl alkylated amino acid residues, based on unique size and charge. Also, several high affinity antibodies have been raised against haptens containing pnitrophenyl moieties, 10 increasing the chances of eliciting an antibody capable of immunoprecipitation. Antibodies raised against hapten 4 are likely to be sequence independent because the binding determinants are relatively distant from the peptide backbone.Utilizing hapten 4, polyclonal antibodies (IgY and IgG) were raised in chickens and rabbits, respectively. 11 To enrich for specific binders, immune antibodies were purified on an affinity column containing immobilized hapten 4. Chicken IgY antibodies (α-3-IgY) performed best shokat@cmp.ucsf The α-3-IgY antibodies only successfully recognized H1 that had been thiophosphorylated and PNBM alky...

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

“…We and others have developed strategies for the capture of thiophosphorylated species (14,15). These strategies rely on the nucleophilicity of the thiophosphate group, which can be modified by reaction with sulfur-selective electrophilic reagents.…”

“…We have recently reported a method for identification of askinase targets by using a monoclonal antibody that recognizes a chemically derivatized thiophosphorylation adduct (14,43). Although the antibody-based approach does not immediately identify the phosphorylation site targeted by an as-kinase, it is complementary to the method reported here because it leads to purification of the entire substrate protein.…”

Generation and characterization of an analog-sensitive PERK allele

“…[28][29][30][31] With this approach, the analog-sensitive kinase uses N 6 -alkylated ATPγS to transfer a thiophosphoryl group to its substrates. The bulky group again confers specificity for the analog-sensitive kinase, and the thiophosphoryl group acts as a label for distinguishing direct substrates from all other phosphorylated proteins in the cell.…”

Identifying Atg1 Substrates: Four Means to an End

“…The first involves the generation of a specific chemical tag on the substrates recognized by the as-PK. 61 In this scheme, the substrates are labeled with A*TPgS, the thio-substituted derivative of the ATP analog. The thioester that is introduced into the candidate substrate is then treated with an alkylating agent to generate a chemical tag that can be recognized immunologically.…”

“…This technique was shown to work for at least one protein kinase, but it remains to be seen how many protein kinases will be able to utilize ATPgS instead of ATP. 61 The second approach combines the analog-specific detection method with a high-throughput analysis. The utility of this approach was demonstrated for the cyclin-dependent kinase, Pho85, from S. cerevisiae.…”