title: Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine Residues.
abstract: Protein phosphorylation on Tyr residues is a key post-translational modification in mammals. In plants, recent studies have identified Tyr-specific protein phosphatase and Tyr-phosphorylated proteins in Arabidopsis by phosphoproteomic screenings, implying that plants have a Tyr phosphorylation signal pathway. However, little is known about the protein kinases (PKs) involved in Tyr phosphorylation in plants. Here, we demonstrate that Arabidopsis calcium-dependent protein kinase (CDPK/CPK)-related PKs (CRKs) have high Tyr-autophosphorylation activity and that they can phosphorylate Tyr residue(s) on substrate proteins in Arabidopsis. To identify PKs for Tyr phosphorylation, we examined the autophosphorylation activity of 759 PKs using an Arabidopsis protein array based on a wheat cell-free system. In total, we identified 38 PKs with Tyr-autophosphorylation activity. The CRK family was a major protein family identified. A cell-free substrate screening revealed that these CRKs phosphorylate β-tubulin (TBB) 2, TBB7, and certain transcription factors (TFs) such as ethylene response factor 13 (ERF13). All five CRKs tested showed Tyr-auto/trans-phosphorylation activity and especially two CRKs, CRK2 and CRK3, showed a high ERF13 Tyr-phosphorylation activity. A cell-based transient expression assay revealed that Tyr(16/)Tyr(207) sites in ERF13 were phosphorylated by CRK3 and that Tyr phosphorylation of endogenous TBBs occurs in CRK2 overexpressing cells. Furthermore, crk2 and crk3 mutants showed a decrease in the Tyr phosphorylation level of TBBs. These results suggest that CRKs have Tyr kinase activity, and these might be one of the major PKs responsible for protein Tyr phosphorylation in Arabidopsis plants.
authors.affiliation: From the Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577 and.
authors.forename: Keiichirou
authors.initials: K
authors.lastname: Nemoto
authors.affiliation: From the Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577 and.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Takemori
authors.affiliation: the Plant Genomic Network Research Team and CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Motoaki
authors.initials: M
authors.lastname: Seki
authors.affiliation: the Gene Discovery Research Group, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, and.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Shinozaki
authors.affiliation: From the Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577 and sawasaki@ehime-u.ac.jp.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005810:Multigene Family; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D014404:Tubulin; D014443:Tyrosine; D011494:Protein Kinases
keywords: Arabidopsis; phosphotyrosine signaling; plant biochemistry; protein array; protein-protein interaction; proteomics