Site-Specific Phosphorylation Profiling of Arabidopsis Proteins by Mass Spectrometry and Peptide Chip Analysis

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 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.

Control of Cell Proliferation, Organ Growth, and DNA Damage Response Operate Independently of Dephosphorylation of the Arabidopsis Cdk1 Homolog CDKA;1

“…However, phosphoproteomics data did also not hint at phosphorylated P-loops of any of the CDKBs (de la Fuente van Bentem et al, 2008;Sugiyama et al, 2008). Thus, we propose here that WEE1 is not a core cell cycle regulator but rather functions as a checkpoint kinase similar to the role of the Mik1 + kinase in S. pombe but targeting other substrates than a PSTAIRE CDK in Arabidopsis (Lundgren et al, 1991;Lee et al, 1994).…”

“…(D) MPF regulation by G2 phase-specific CDK inhibitors (CKI*): a stoichiometrically acting CKI can delay MPF activation instead of inhibitory phosphorylations onto CDK-cyclin complexes. et al, 2007; de la Fuente van Bentem et al, 2008;Sugiyama et al, 2008). However, these are negative data, and caution is required in interpreting the absence of specific phosphopeptides.…”

Regulation of Leaf Starch Degradation by Abscisic Acid Is Important for Osmotic Stress Tolerance in Plants

“…Conversely, there is no evidence for redox regulation of BAM3, and the enzyme has optimum activity at less alkaline pH (Santelia et al, 2015;Monroe et al, 2014). Furthermore, large-scale site-specific phosphorylation profiling of Arabidopsis proteins revealed the presence of one or more phosphorylation sites on both BAM1 and AMY3 proteins (de la Fuente van Bentem et al, 2008;Reiland et al, 2009;Xue et al, 2013;Heazlewood et al, 2008). Given the rapid activation of leaf starch degradation in response to the osmotic stress, it is likely that posttranslational modifications such as protein phosphorylation or redox regulation played a major role in activating BAM1 and AMY3 in the light.…”

The gateway to chloroplast: re-defining the function of chloroplast receptor proteins

“…In agreement with the concept that reversible protein phosphorylation is central to the regulation of most aspects of cell function (Johnson , 2009 ), many disordered regions present in proteins are indeed regulated by phosphorylation (Dyson and Wright , 2005 ). Phosphoproteomic profiling of A. thaliana proteins from several independent studies revealed that the A-domain as well as the full-length Toc159 is phosphorylated (de la Fuente van Bentem et al , 2008 ;Reiland et al , 2009 ). Similarly, cell fractionation followed by in vitro phosphospecific staining further demonstrated that fulllength Toc159 and the free A-domain were indeed both phosphoproteins (Agne et al , 2010 ).…”