Site-specific phosphorylation profiling of arabidopsis proteins by mass spectrometry and peptide chip analysis

Sergio De La Fuente Van Bentem*, Dorothea Anrather, Ilse Dohnal, Elisabeth Roitinger, Edina Csaszar, Jos Joore, Joshua Buijnink, Alessandro Carreri, Celine Forzani, Zdravko J. Lorkovic, Andrea Barta, David Lecourieux, Andreas Verhounig, Claudia Jonak, Heribert Hirt

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

111 Scopus citations

Abstract

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear- enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H 2O2-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H 2O2- induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H 2O2. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.

Original languageEnglish (US)
Pages (from-to)2458-2470
Number of pages13
JournalJournal of Proteome Research
Volume7
Issue number6
DOIs
StatePublished - Jun 1 2008

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Fingerprint Dive into the research topics of 'Site-specific phosphorylation profiling of arabidopsis proteins by mass spectrometry and peptide chip analysis'. Together they form a unique fingerprint.

Cite this