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*

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 onedimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine/threonine-phosphorylated proteins including drebrin 1, -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. Molecular & Cellular Proteomics 1:517–527, 2002. Signal transduction events involve transmission and amplification of signals from transmembrane receptors to the nucleus. One major conduit of information is mediated via reversible phosphorylation of proteins (1–3). In eukaryotes, phosphorylation occurs chiefly on serine, threonine, and tyrosine residues. Phosphorylation on serines and threonines is far more abundant than that on tyrosine residues. Indeed, the ratio of phosphorylation on phosphoserine:phosphothreonine:phosphotyrosine is 1800:200:1 in vertebrates (4). The overall level of phosphorylation is regulated by an interplay between the activities of protein kinases, as well as protein phosphatases within cells. Although tyrosine phosphorylation is far less frequent than serine/threonine phosphorylation in cells, it has been studied quite intensively. One of the major reasons for this is the availability of excellent antibodies that recognize phosphorylated tyrosine residues in immunoprecipitation, as well as Western blotting experiments. In fact, detection of a protein by these antibodies generally constitutes proof that the protein is tyrosine-phosphorylated. Antibodies that specifically recognize serine/threonine-phosphorylated proteins have become available in recent years. However, a major drawback is that they cannot be used for enrichment purposes, because they do not work in immunoprecipitation experiments. Although strategies such as immobilized metal affinity chromatography have been developed for selective enrichment of phosphorylated peptides (5–8), their use to enrich for phosphorylated proteins has not yet been demonstrated. Other strategies to enrich for serine/threonine-phosphorylated proteins have been published, but they involve a series of chemical modifications that can result in loss of proteins (9–11). In this report, we have tested several antibodies for their ability to immunoprecipitate and Western blot serine/threonine-phosphorylated proteins. We show that some of these antibodies can be used to specifically immunoprecipitate proteins phosphorylated on serine/threonine residues. We performed a large scale purification of proteins whose phosphorylation was induced by treatment of cells with calyculin A, a phosphatase inhibitor. Mass spectrometry has emerged as From the ‡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, and ‡‡Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 and Brigham and Women’s Hospital, Boston, Massachusetts 02115 Received, February 18, 2002, and in revised form, July 5, 2002 Published, MCP Papers in Press, July 22, 2002, DOI 10.1074/mcp.M200010-MCP200 Research