title: Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases.
abstract: BACKGROUND
  Mitogen-activated protein kinase (MPK) cascades are important to cellular signaling in eukaryotes. They regulate growth, development and the response to environmental challenges. MPK cascades function via reversible phosphorylation of cascade components, MEKK, MEK, and MPK, but also by MPK substrate phosphorylation. Using mass spectrometry, we previously identified many in vivo MPK3 and MPK6 substrates in Arabidopsis thaliana, and we disclosed their phosphorylation sites.
  
  
  RESULTS
  We verified phosphorylation of several of our previously identified MPK3/6 substrates using a nonradioactive in vitro labeling assay. We engineered MPK3, MPK4, and MPK6 to accept bio-orthogonal ATPγS analogs for thiophosphorylating their appropriate substrate proteins. Subsequent alkylation of the thiophosphorylated amino acid residue(s) allows immunodetection using thiophosphate ester-specific antibodies. Site-directed mutagenesis of amino acids confirmed the protein substrates' site-specific phosphorylation by MPK3 and MPK6. A combined assay with MPK3, MPK6, and MPK4 revealed substrate specificity of the individual kinases.
  
  
  CONCLUSIONS
  Our work demonstrates that the in vitro-labeling assay represents an effective, specific and highly sensitive test for determining kinase-substrate relationships.
authors.affiliation: Department of Plant Physiology, Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, 52056, Germany. franz.leissing@rwth-aachen.de.
authors.forename: Franz
authors.initials: F
authors.lastname: Leissing
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan. nomoto.mika@a.mbox.nagoya-u.ac.jp.
authors.forename: Mika
authors.initials: M
authors.lastname: Nomoto
authors.affiliation: Department of Biotechnology, Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, 52056, Germany. m.bocola@biotec.rwth-aachen.de.
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.affiliation: Department of Biotechnology, Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, 52056, Germany. u.schwaneberg@biotec.rwth-aachen.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan. ytada@gene.nagoya-u.ac.jp.|The Center for Gene Research, Division of Biological Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan. ytada@gene.nagoya-u.ac.jp.
authors.forename: Yasuomi
authors.initials: Y
authors.lastname: Tada
authors.affiliation: Department of Plant Physiology, Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, 52056, Germany. uwe.conrath@bio3.rwth-aachen.de.
authors.forename: Uwe
authors.initials: U
authors.lastname: Conrath
authors.affiliation: Department of Plant Physiology, Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, 52056, Germany. beckers@bio3.rwth-aachen.de.
authors.forename: Gerold J M
authors.initials: GJ
authors.lastname: Beckers
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; C458516:AtMPK3 protein, Arabidopsis; C436293:MPK6 protein, Arabidopsis; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases