Bio-orthogonal affinity purification of direct kinase substrates

Abstract

Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signaling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. Herein, we describe a chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase. The kinase of interest is engineered to transfer a phosphorothioate moiety to phosphoacceptor hydroxyl groups on direct substrates. In a second nonenzymatic step, the introduced phosphorothioate is alkylated with p-nitrobenzylmesylate (PNBM). Antibodies directed against the alkylated phosphorothioate epitope recognize these labeled substrates, but not alkylation products of other cellular nucleophiles. This strategy is demonstrated with Cdk1/cyclinB substrates using ELISA, western blotting, and immunoprecipitation in the context of whole cell lysates.

Figure 1

Recognition determinants for α-3-IgY immunoreactivity measured by western blotting; 25 ng of Swe1 or Swe1-P^S was treated with DMSO (lanes 1 and 3) or 2.5 mM PNBM in DMSO (lanes 2 and 4); 15 μg of WCL was treated with DMSO (lane 5), 2.5 mM PNBM in DMSO (lane 6), and 25 ng of Swe1-P^S plus 2.5 mM PNBM in DMSO (lane 7). Lanes 8 and 9 show coomassie staining of samples identical to 6 and 7, respectively.

Figure 2

Immunoprecipitation of Rh-H1-P^S+PNBM measured by fluorescence of the SDS–PAGE resolved immunoprecipitates. WCL* indicates treatment with PNBM. Lanes 1–3 were treated with α-3-IgY sepharose, and lanes 4–5 with preimmune IgY sepharose.

Scheme 1

Tandem Approach for Creating Bio-orthogonal Affinity Tagged Kinase Substrates (Ser R = H, Thr R = Me)

Scheme 2

Immunoaffinity Purification