Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism.
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.
de la Fuente van Bentem
Sergio
S
Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr Bohr-Gasse 9, 1030 Vienna, Austria. sergio.de.la.fuente.van.bentem@univie.ac.at
Anrather
Dorothea
D
Roitinger
Elisabeth
E
Djamei
Armin
A
Hufnagl
Thomas
T
Barta
Andrea
A
Csaszar
Edina
E
Dohnal
Ilse
I
Lecourieux
David
D
Hirt
Heribert
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D018749:RNA, Plant / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000068103:Serine-Arginine Splicing Factors
D029681:Arabidopsis Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D018749:RNA, Plant; D016601:RNA-Binding Proteins; C103793:RSp31 protein, Arabidopsis; D000068103:Serine-Arginine Splicing Factors; D017346:Protein Serine-Threonine Kinases