Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis.
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.
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. delafus3@univie.ac.at
Anrather
Dorothea
D
Dohnal
Ilse
I
Roitinger
Elisabeth
E
Csaszar
Edina
E
Joore
Jos
J
Buijnink
Joshua
J
Carreri
Alessandro
A
Forzani
Celine
C
Lorkovic
Zdravko J
ZJ
Barta
Andrea
A
Lecourieux
David
D
Verhounig
Andreas
A
Jonak
Claudia
C
Hirt
Heribert
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D060565:Argonaute Proteins; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D006005:Phosphorylases / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D040081:Protein Array Analysis / Q000379:methods*; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods
D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D018844:Cyclin-Dependent Kinases; D020690:GTPase-Activating Proteins; D038362:Glycogen Synthase Kinase 3; D006861:Hydrogen Peroxide; D020928:Mitogen-Activated Protein Kinases; D004676:Myelin Basic Protein; D010455:Peptides; D010750:Phosphoproteins; D006005:Phosphorylases; D011494:Protein Kinases; C117457:AGO1 protein, Arabidopsis; C511291:At1g15750 protein, Arabidopsis; C085936:ATSK11 protein, Arabidopsis