Published online 17 July 2006
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism
Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna Dr Bohr-Gasse 9, 1030 Vienna, Austria 1 Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna Dr Bohr-Gasse 9, 1030 Vienna, Austria 2 Research Institute of Molecular Pathology, Max F. Perutz Laboratories, Medical University of Vienna Dr Bohr-Gasse 9, 1030 Vienna, Austria 3 Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna Dr Bohr-Gasse 9, 1030 Vienna, Austria
*To whom correspondence should be addressed. Tel: +43 1 4277 54622; Fax: +43 1 4277 9546; Email: sergio.de.la.fuente.van.bentem{at}univie.ac.at
Received March 27, 2006. Revised May 25, 2006. Accepted May 30, 2006.
Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.
Present addresses: Elisabeth Roitinger, Christian-Doppler-Laboratory for Proteome Analysis, Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Dr Bohr-Gasse 9, 1030 Vienna, Austria
David Lecourieux, UMR CNRS 6161, Bâtiment de Botanique, 40 avenue du recteur Pineau, F-86022, Poitiers cedex, France
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