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Originally published In Press as doi:10.1074/mcp.M200010-MCP200 on July 22, 2002.
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Molecular & Cellular Proteomics 1:517-527, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

A Mass Spectrometry-based Proteomic Approach for Identification of Serine/Threonine-phosphorylated Proteins by Enrichment with Phospho-specific Antibodies

Identification of a Novel Protein, Frigg, as a Protein Kinase A Substrate*

Mads Grønborg{ddagger},§, Troels Zakarias Kristiansen{ddagger}, Allan Stensballe{ddagger}, Jens S. Andersen{ddagger}, Osamu Ohara, Matthias Mann{ddagger},||, Ole Nørregaard Jensen{ddagger},** and Akhilesh Pandey{ddagger}{ddagger},§§

{ddagger} Department of Biochemistry and Molecular Biology, Center for Experimental Bioinformatics, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
Kazusa DNA Research Institute and RIKEN Research Center for Allergy and Immunology, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan
{ddagger}{ddagger} Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 and Brigham and Women’s Hospital, Boston, Massachusetts 02115

Although proteins phosphorylated on tyrosine residues can be enriched by immunoprecipitation with anti-phosphotyrosine antibodies, it has been difficult to identify proteins that are phosphorylated on serine/threonine residues because of lack of immunoprecipitating antibodies. In this report, we describe several antibodies that recognize phosphoserine/phosphothreonine-containing proteins by Western blotting. Importantly, these antibodies can be used to enrich for proteins phosphorylated on serine/threonine residues by immunoprecipitation, as well. Using these antibodies, we have immunoprecipitated proteins from untreated cells or those treated with calyculin A, a serine/threonine phosphatase inhibitor. Mass spectrometry-based analysis of bands from one-dimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine/threonine-phosphorylated proteins including drebrin 1, {alpha}-actinin 4, and filamin-1. We also identified a protein, poly(A)-binding protein 2, which was previously not known to be phosphorylated, in addition to a novel protein without any obvious domains that we designate as Frigg. Frigg is widely expressed and was demonstrated to be a protein kinase A substrate in vitro. We identified several in vivo phosphorylation sites by tandem mass spectrometry using Frigg protein immunoprecipitated from cells. Our method should be applicable as a generic strategy for enrichment and identification of serine/threonine-phosphorylated substrates in signal transduction pathways.

|| To whom correspondence may be addressed. Tel.: 45-65502364; Fax: 45-65933929; E-mail: mann{at}bmb.sdu.dk

** To whom correspondence may be addressed. Tel.: 45-65502368; Fax: 45-65932661; E-mail: jenseno{at}bmb.sdu.dk


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