Comparative analysis of phytohormone-responsive phosphoproteins in Arabidopsis thaliana using TiO2-phosphopeptide enrichment and mass accuracy precursor alignment.

Protein phosphorylation/dephosphorylation is a central post-translational modification in plant hormone signaling, but little is known about its extent and function. Although pertinent protein kinases and phosphatases have been predicted and identified for a variety of hormone responses, classical biochemical approaches have so far revealed only a few candidate proteins and even fewer phosphorylation sites. Here we performed a global quantitative analysis of the Arabidopsis phosphoproteome in response to a time course of treatments with various plant hormones using phosphopeptide enrichment and subsequent mass accuracy precursor alignment (MAPA). The use of three time points, 1, 3 and 6 h, in combination with five phytohormone treatments, abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellic acid (GA), jasmonic acid (JA) and kinetin, resulted in 324,000 precursor ions from 54 LC-Orbitrap-MS analyses quantified and aligned in a data matrix with the dimension of 6000 x 54 using the ProtMax algorithm. To dissect the phytohormone responses, multivariate principal/independent components analysis was performed. In total, 152 phosphopeptides were identified as differentially regulated; these phosphopeptides are involved in a wide variety of signaling pathways. New phosphorylation sites were identified for ABA response element binding factors that showed a specific increase in response to ABA. New phosphorylation sites were also found for RLKs and auxin transporters. We found that different hormones regulate distinct amino acid residues of members of the same protein families. In contrast, tyrosine phosphorylation of the G alpha subunit appeared to be a common response for multiple hormones, demonstrating global cross-talk among hormone signaling pathways.

[1]  E. Chevet,et al.  Calnexin family members as modulators of genetic diseases. , 1999, Seminars in cell & developmental biology.

[2]  Xiangdong Fu,et al.  Auxin promotes Arabidopsis root growth by modulating gibberellin response , 2003, Nature.

[3]  Elliot M. Meyerowitz,et al.  Antagonistic Regulation of PIN Phosphorylation by PP2A and PINOID Directs Auxin Flux , 2007, Cell.

[4]  P. Roepstorff,et al.  Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns* , 2005, Molecular & Cellular Proteomics.

[5]  W. Frommer,et al.  Temporal Analysis of Sucrose-induced Phosphorylation Changes in Plasma Membrane Proteins of Arabidopsis*S , 2007, Molecular & Cellular Proteomics.

[6]  Hironori Takasaki,et al.  Comprehensive phosphoproteome analysis in rice and identification of phosphoproteins responsive to different hormones/stresses. , 2005, Journal of proteome research.

[7]  Alan M. Jones,et al.  The beta-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes. , 2003, The Plant cell.

[8]  K. Shinozaki,et al.  Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[9]  K. Murthy,et al.  Gi-coupled receptors mediate phosphorylation of CPI-17 and MLC20 via preferential activation of the PI3K/ILK pathway. , 2006, The Biochemical journal.

[10]  Adrian D Hegeman,et al.  A Quantitative Analysis of Arabidopsis Plasma Membrane Using Trypsin-catalyzed 18O Labeling * S , 2006, Molecular & Cellular Proteomics.

[11]  G. Bolbach,et al.  Protein Tyrosine Kinases and Protein Tyrosine Phosphatases Are Involved in Abscisic Acid-Dependent Processes in Arabidopsis Seeds and Suspension Cells1 , 2008, Plant Physiology.

[12]  R. Reski,et al.  Rapid alteration of the phosphoproteome in the moss Physcomitrella patens after cytokinin treatment. , 2006, Journal of proteome research.

[13]  K. Shinozaki,et al.  ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis. , 2002, Plant & cell physiology.

[14]  I. Hwang,et al.  Arabidopsis Calcium-Dependent Protein Kinase AtCPK32 Interacts with ABF4, a Transcriptional Regulator of Abscisic Acid-Responsive Gene Expression, and Modulates Its Activity1 , 2005, Plant Physiology.

[15]  Jia Li,et al.  BAK1, an Arabidopsis LRR Receptor-like Protein Kinase, Interacts with BRI1 and Modulates Brassinosteroid Signaling , 2002, Cell.

[16]  Y. Hannun,et al.  Regulation of volume‐activated chloride channels by P‐glycoprotein: phosphorylation has the final say! , 2000, The Journal of physiology.

[17]  Y. Matsubayashi,et al.  An LRR Receptor Kinase Involved in Perception of a Peptide Plant Hormone, Phytosulfokine , 2002, Science.

[18]  Joost T. van Dongen,et al.  A rapid approach for phenotype‐screening and database independent detection of cSNP/protein polymorphism using mass accuracy precursor alignment , 2008, Proteomics.

[19]  K. Mockaitis,et al.  Protein phosphorylation in the delivery of and response to auxin signals , 2002, Plant Molecular Biology.

[20]  K. Shinozaki,et al.  Leucine-Rich Repeat Receptor-Like Kinase1 Is a Key Membrane-Bound Regulator of Abscisic Acid Early Signaling in Arabidopsisw⃞ , 2005, The Plant Cell Online.

[21]  Joachim Selbig,et al.  ProMEX: a mass spectral reference database for proteins and protein phosphorylation sites , 2007, BMC Bioinformatics.

[22]  K. Mikoshiba,et al.  Activation of the G protein Gq/11 through tyrosine phosphorylation of the alpha subunit. , 1997, Science.

[23]  S. Komatsu,et al.  Identification of phosphoproteins regulated by gibberellin in rice leaf sheath , 2005, Plant Molecular Biology.

[24]  F. Baluška,et al.  Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin B. , 2006, The Plant journal : for cell and molecular biology.

[25]  R. Deshaies.  SCF and Cullin/Ring H2-based ubiquitin ligases. , 1999, Annual review of cell and developmental biology.

[26]  Masahiko Furutani,et al.  The gene MACCHI-BOU 4/ENHANCER OF PINOID encodes a NPH3-like protein and reveals similarities between organogenesis and phototropism at the molecular level , 2007, Development.

[27]  T. Pawson,et al.  Structural Basis for Autoinhibition of the EphB2 Receptor Tyrosine Kinase by the Unphosphorylated Juxtamembrane Region , 2001, Cell.

[28]  Wei-Hua Wu,et al.  A G Protein-Coupled Receptor Is a Plasma Membrane Receptor for the Plant Hormone Abscisic Acid , 2007, Science.

[29]  P. Pandolfi,et al.  Phosphorylation-dependent regulation of cytosolic localization and oncogenic function of Skp2 by Akt/PKB , 2009, Nature Cell Biology.

[30]  M. Palmgren,et al.  The Plant Plasma Membrane Ca2+ Pump ACA8 Contains Overlapping as Well as Physically Separated Autoinhibitory and Calmodulin-binding Domains* , 2006, Journal of Biological Chemistry.

[31]  J. Vázquez-Prado,et al.  G protein-coupled receptor cross-talk: pivotal roles of protein phosphorylation and protein-protein interactions. , 2003, Cellular signalling.

[32]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[33]  Dirk Walther,et al.  Plant phosphoproteomics: An update , 2009, Proteomics.

[34]  Sarah M Assmann,et al.  The Arabidopsis Putative G Protein–Coupled Receptor GCR1 Interacts with the G Protein α Subunit GPA1 and Regulates Abscisic Acid Signaling , 2004, The Plant Cell Online.

[35]  A. Diévart,et al.  LRR-containing receptors regulating plant development and defense , 2003, Development.

[36]  D. Van Der Straeten,et al.  The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristem-identity genes , 2007, Proceedings of the National Academy of Sciences.

[37]  O. Fiehn,et al.  Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co‐regulation in biochemical networks , 2004, Proteomics.

[38]  Alan M. Jones,et al.  GCR1 Can Act Independently of Heterotrimeric G-Protein in Response to Brassinosteroids and Gibberellins in Arabidopsis Seed Germination1[w] , 2004, Plant Physiology.

[39]  M. Mann,et al.  Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks , 2006, Cell.

[40]  M. Akabas.  Cystic Fibrosis Transmembrane Conductance Regulator , 2000, The Journal of Biological Chemistry.

[41]  Joachim Selbig,et al.  PhosPhAt: a database of phosphorylation sites in Arabidopsis thaliana and a plant-specific phosphorylation site predictor , 2007, Nucleic Acids Res..

[42]  I. Pastan,et al.  Biochemical, cellular, and pharmacological aspects of the multidrug transporter. , 1999, Annual review of pharmacology and toxicology.

[43]  M. Carlson,et al.  Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. , 1996, Genes & development.