Bio-orthogonal affinity purification of direct kinase substrates.
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.
Allen
Jasmina J
JJ
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, USA.
Lazerwith
Scott E
SE
Shokat
Kevan M
KM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism; D008698:Mesylates / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D000345:Affinity Labels; D018797:Cell Cycle Proteins; D006657:Histones; C016512:IgY; D018796:Immunoconjugates; D007074:Immunoglobulin G; D007136:Immunoglobulins; D008698:Mesylates; D029701:Saccharomyces cerevisiae Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C099685:SWE1 protein, S cerevisiae; D011505:Protein-Tyrosine Kinases; D016203:CDC2 Protein Kinase