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

Sergio De La Fuente Van Bentem; Dorothea Anrather; Elisabeth Roitinger; Armin Djamei; Thomas Hufnagl; Andrea Barta; Edina Csaszar; Ilse Dohnal; David Lecourieux; Heribert Hirt

doi:10.1093/nar/gkl429

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

Sergio De La Fuente Van Bentem^*, Dorothea Anrather, Elisabeth Roitinger, Armin Djamei, Thomas Hufnagl, Andrea Barta, Edina Csaszar, Ilse Dohnal, David Lecourieux, Heribert Hirt

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

113 Scopus citations

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 (LCMS/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.

Original language	English (US)
Pages (from-to)	3267-3278
Number of pages	12
Journal	NUCLEIC ACIDS RESEARCH
Volume	34
Issue number	11
DOIs	https://doi.org/10.1093/nar/gkl429
State	Published - 2006
Externally published	Yes

Bibliographical note

Funding Information:
We thank Andriy Belokurov for assistance with the Arabidopsis cell culture. This work was supported by the Austrian Science Foundation, the Vienna Science and Technology Fund and the European Union. Funding to pay the Open Access publication charges for this article was provided by the Vienna Science and Technology Fund.

ASJC Scopus subject areas

Genetics

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title = "Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism",

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 (LCMS/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.",

author = "{De La Fuente Van Bentem}, Sergio and Dorothea Anrather and Elisabeth Roitinger and Armin Djamei and Thomas Hufnagl and Andrea Barta and Edina Csaszar and Ilse Dohnal and David Lecourieux and Heribert Hirt",

note = "Funding Information: We thank Andriy Belokurov for assistance with the Arabidopsis cell culture. This work was supported by the Austrian Science Foundation, the Vienna Science and Technology Fund and the European Union. Funding to pay the Open Access publication charges for this article was provided by the Vienna Science and Technology Fund.",

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AU - De La Fuente Van Bentem, Sergio

AU - Anrather, Dorothea

AU - Roitinger, Elisabeth

AU - Djamei, Armin

AU - Hufnagl, Thomas

AU - Barta, Andrea

AU - Csaszar, Edina

AU - Dohnal, Ilse

AU - Lecourieux, David

AU - Hirt, Heribert

N1 - Funding Information: We thank Andriy Belokurov for assistance with the Arabidopsis cell culture. This work was supported by the Austrian Science Foundation, the Vienna Science and Technology Fund and the European Union. Funding to pay the Open Access publication charges for this article was provided by the Vienna Science and Technology Fund.

PY - 2006

Y1 - 2006

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

Abstract

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