Publication Information
de la Fuente van Bentem et al., 2008
No external accession available
Abstract
J Proteome Res. 2008 Jun;7(6):2458-70. doi: 10.1021/pr8000173. Epub 2008 Apr 24.
Site-specific phosphorylation profiling of Arabidopsis proteins by mass
spectrometry and peptide chip analysis.
de la Fuente van Bentem S(1), Anrather D, Dohnal I, Roitinger E, Csaszar E,
Joore J, Buijnink J, Carreri A, Forzani C, Lorkovic ZJ, Barta A, Lecourieux D,
Verhounig A, Jonak C, Hirt H.
Author information:
(1)Department of Plant Molecular Biology, Max F. Perutz Laboratories, University
of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria. delafus3@univie.ac.at
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 2O
2-treated Arabidopsis cells. Different peptide phosphorylation profiles
indicated the presence of overlapping but distinct PK activities in cytosolic
and nuclear compartments. Among different H 2O 2-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 2O 2. 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.
DOI: 10.1021/pr8000173
PMID: 18433157 [Indexed for MEDLINE]
