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.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, U.S. Gov't, P.H.S.
MeSH terms
-
Adenosine Triphosphate / analogs & derivatives*
-
Adenosine Triphosphate / chemistry
-
Adenosine Triphosphate / metabolism
-
Affinity Labels / chemistry*
-
Affinity Labels / metabolism
-
CDC2 Protein Kinase / metabolism*
-
Cell Cycle Proteins / chemistry*
-
Cell Cycle Proteins / isolation & purification
-
Cell Cycle Proteins / metabolism
-
HeLa Cells
-
Histones / chemistry*
-
Histones / isolation & purification
-
Histones / metabolism
-
Humans
-
Immunoconjugates / chemistry*
-
Immunoconjugates / metabolism
-
Immunoglobulin G / chemistry
-
Immunoglobulin G / metabolism
-
Immunoglobulins / chemistry
-
Immunoglobulins / metabolism
-
Mesylates / chemistry
-
Protein-Tyrosine Kinases / chemistry*
-
Protein-Tyrosine Kinases / isolation & purification
-
Protein-Tyrosine Kinases / metabolism
-
Saccharomyces cerevisiae Proteins / chemistry*
-
Saccharomyces cerevisiae Proteins / isolation & purification
-
Saccharomyces cerevisiae Proteins / metabolism
-
Substrate Specificity
Substances
-
Affinity Labels
-
Cell Cycle Proteins
-
Histones
-
IgY
-
Immunoconjugates
-
Immunoglobulin G
-
Immunoglobulins
-
Mesylates
-
Saccharomyces cerevisiae Proteins
-
adenosine 5'-O-(3-thiotriphosphate)
-
Adenosine Triphosphate
-
SWE1 protein, S cerevisiae
-
Protein-Tyrosine Kinases
-
CDC2 Protein Kinase