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 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.
Grønborg
Mads
M
Department of Biochemistry and Molecular Biology, Center for Experimental Bioinformatics, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
Kristiansen
Troels Zakarias
TZ
Stensballe
Allan
A
Andersen
Jens S
JS
Ohara
Osamu
O
Mann
Matthias
M
Jensen
Ole Nørregaard
ON
Pandey
Akhilesh
A
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D002460:Cell Line; D003285:Contractile Proteins / Q000032:analysis / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D064448:Filamins; D006801:Humans; D008387:Marine Toxins; D008840:Microfilament Proteins / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D010080:Oxazoles / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis* / Q000737:chemistry; D010769:Phosphothreonine / Q000032:analysis* / Q000737:chemistry; D011233:Precipitin Tests; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D003285:Contractile Proteins; D004791:Enzyme Inhibitors; D064448:Filamins; D008387:Marine Toxins; D008840:Microfilament Proteins; D010080:Oxazoles; D010446:Peptide Fragments; D010750:Phosphoproteins; D010769:Phosphothreonine; D010768:Phosphoserine; C059041:calyculin A