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PMID: 16807317 Published · epublish English Evaluation Study Journal Article Research Support, Non-U.S. Gov't

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism.

Nucleic acids research ·Vol. 34 ·No. 11 ·2006-00-00 ·Pages 3267-78

de la Fuente van Bentem S, Anrather D, Roitinger E, Djamei A, Hufnagl T, Barta A, Csaszar E, Dohnal I, Lecourieux D, Hirt H

Abstract

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.

MeSH Terms
Amino Acid Motifs Amino Acid Sequence Arabidopsis/enzymology,metabolism Arabidopsis Proteins/chemistry,metabolism Cell Nucleus/metabolism Chromatography, Affinity Cytosol/metabolism Mass Spectrometry Molecular Sequence Data Phosphopeptides/chemistry,isolation & purification Phosphoproteins/chemistry,metabolism Phosphorylation Protein Isoforms/metabolism Protein Serine-Threonine Kinases/metabolism Proteomics/methods RNA Splicing RNA, Messenger/metabolism RNA, Plant/metabolism RNA-Binding Proteins/metabolism Sequence Homology, Amino Acid Serine-Arginine Splicing Factors
Chemicals
Arabidopsis Proteins Phosphopeptides Phosphoproteins Protein Isoforms RNA, Messenger RNA, Plant RNA-Binding Proteins RSp31 protein, Arabidopsis Serine-Arginine Splicing Factors Protein Serine-Threonine Kinases
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
de la Fuente van Bentem Sergio
Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr Bohr-Gasse 9, 1030 Vienna, Austria. [email protected]
Anrather Dorothea
Roitinger Elisabeth
Djamei Armin
Hufnagl Thomas
Barta Andrea
Csaszar Edina
Dohnal Ilse
Lecourieux David
Hirt Heribert
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkl429
Email
[email protected]
Published
2006-00-00
Epub
2006-00-28
Pages
3267-78
Language
English
Region
England
NLM ID
0411011
PMCID
PMC1904105
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/gkl429 PMC Full Text View journal details
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