HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: M200010-MCP200v1 1/7/517    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Grønborg, M. Articles by Pandey, A. Search for Related Content PubMed PubMed Citation Articles by Grønborg, M. Articles by Pandey, A. Social Bookmarking                      What's this?

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^,, Troels Zakarias Kristiansen, Allan Stensballe, Jens S. Andersen, Osamu Ohara^¶, Matthias Mann^,||, Ole Nørregaard Jensen^,** and Akhilesh Pandey^,

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
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, -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.: 45-65502368; Fax: 45-65932661; E-mail: jenseno{at}bmb.sdu.dk

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. Lin, Z. Xie, H. Zhu, and J. Qian Understanding protein phosphorylation on a systems level Briefings in Functional Genomics, January 7, 2010; (2010) elp045v1. [Abstract] [Full Text] [PDF]

				E. Lecuona, A. Minin, H. E. Trejo, J. Chen, A. P. Comellas, H. Sun, D. Grillo, O. E. Nekrasova, L. C. Welch, I. Szleifer, et al. Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells J. Cell Sci., November 1, 2009; 122(21): 3915 - 3922. [Abstract] [Full Text] [PDF]

				D. Razafsky and D. Hodzic Bringing KASH under the SUN: the many faces of nucleo-cytoskeletal connections J. Cell Biol., August 24, 2009; 186(4): 461 - 472. [Abstract] [Full Text] [PDF]

				K. Blackburn and M. B. Goshe Challenges and strategies for targeted phosphorylation site identification and quantification using mass spectrometry analysis Briefings in Functional Genomics, March 1, 2009; 8(2): 90 - 103. [Abstract] [Full Text] [PDF]

				S. P. Mirza and M. Olivier Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry Physiol Genomics, October 8, 2008; 33(1): 3 - 11. [Abstract] [Full Text] [PDF]

				C. Jorgensen and R. Linding Directional and quantitative phosphorylation networks Briefings in Functional Genomics, February 12, 2008; (2008) eln001v1. [Abstract] [Full Text] [PDF]

				X. Li, J. W. Kim, M. Gronborg, H. Urlaub, M. D. Lane, and Q.-Q. Tang Role of cdk2 in the sequential phosphorylation/activation of C/EBPbeta during adipocyte differentiation PNAS, July 10, 2007; 104(28): 11597 - 11602. [Abstract] [Full Text] [PDF]

				N. Sugiyama, T. Masuda, K. Shinoda, A. Nakamura, M. Tomita, and Y. Ishihama Phosphopeptide Enrichment by Aliphatic Hydroxy Acid-modified Metal Oxide Chromatography for Nano-LC-MS/MS in Proteomics Applications Mol. Cell. Proteomics, June 1, 2007; 6(6): 1103 - 1109. [Abstract] [Full Text] [PDF]

				J. A. Schumacher, D. K. Crockett, K. S.J. Elenitoba-Johnson, and M. S. Lim Evaluation of Enrichment Techniques for Mass Spectrometry: Identification of Tyrosine Phosphoproteins in Cancer Cells J. Mol. Diagn., April 1, 2007; 9(2): 169 - 177. [Abstract] [Full Text] [PDF]

				J. W. Kehoe, N. Velappan, M. Walbolt, J. Rasmussen, D. King, J. Lou, K. Knopp, P. Pavlik, J. D. Marks, C. R. Bertozzi, et al. Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context Mol. Cell. Proteomics, December 1, 2006; 5(12): 2350 - 2363. [Abstract] [Full Text] [PDF]

				M. Livingstone, H. Ruan, J. Weiner, K. R. Clauser, P. Strack, S. Jin, A. Williams, H. Greulich, J. Gardner, M. Venere, et al. Valosin-Containing Protein Phosphorylation at Ser784 in Response to DNA Damage Cancer Res., September 1, 2005; 65(17): 7533 - 7540. [Abstract] [Full Text] [PDF]

				F.L.C. Moseley, K.N. Jha, L. Bjorndahl, I.A. Brewis, S.J. Publicover, C.L.R. Barratt, and L. Lefievre Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation Mol. Hum. Reprod., July 1, 2005; 11(7): 523 - 529. [Abstract] [Full Text] [PDF]

				A. T. Parent, N. Y. Barnes, Y. Taniguchi, G. Thinakaran, and S. S. Sisodia Presenilin Attenuates Receptor-Mediated Signaling and Synaptic Function J. Neurosci., February 9, 2005; 25(6): 1540 - 1549. [Abstract] [Full Text] [PDF]

				R. Amanchy, D. E. Kalume, and A. Pandey Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) for Studying Dynamics of Protein Abundance and Posttranslational Modifications Sci. Signal., January 18, 2005; 2005(267): pl2 - pl2. [Abstract] [Full Text] [PDF]

				S. A. Beausoleil, M. Jedrychowski, D. Schwartz, J. E. Elias, J. Villen, J. Li, M. A. Cohn, L. C. Cantley, and S. P. Gygi Large-scale characterization of HeLa cell nuclear phosphoproteins PNAS, August 17, 2004; 101(33): 12130 - 12135. [Abstract] [Full Text] [PDF]

				L. Al-Khalili, O. Kotova, H. Tsuchida, I. Ehren, E. Feraille, A. Krook, and A. V. Chibalin ERK1/2 Mediates Insulin Stimulation of Na,K-ATPase by Phosphorylation of the {alpha}-Subunit in Human Skeletal Muscle Cells J. Biol. Chem., June 11, 2004; 279(24): 25211 - 25218. [Abstract] [Full Text] [PDF]

				C. O'Flaherty, E. de Lamirande, and C. Gagnon Phosphorylation of the Arginine-X-X-(Serine/Threonine) motif in human sperm proteins during capacitation: modulation and protein kinase A dependency Mol. Hum. Reprod., May 1, 2004; 10(5): 355 - 363. [Abstract] [Full Text] [PDF]

				T. S. Nuhse, A. Stensballe, O. N. Jensen, and S. C. Peck Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry Mol. Cell. Proteomics, November 1, 2003; 2(11): 1234 - 1243. [Abstract] [Full Text] [PDF]

				K. Machida, B. J. Mayer, and P. Nollau Profiling the Global Tyrosine Phosphorylation State Mol. Cell. Proteomics, April 1, 2003; 2(4): 215 - 233. [Abstract] [Full Text] [PDF]

				G. S. Baillie, A. Sood, I. McPhee, I. Gall, S. J. Perry, R. J. Lefkowitz, and M. D. Houslay beta -Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta -adrenoceptor switching from Gs to Gi PNAS, February 4, 2003; 100(3): 940 - 945. [Abstract] [Full Text] [PDF]

				L. Wells, K. Vosseller, R. N. Cole, J. M. Cronshaw, M. J. Matunis, and G. W. Hart Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications Mol. Cell. Proteomics, October 1, 2002; 1(10): 791 - 804. [Abstract] [Full Text] [PDF]