title: Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis.
abstract: Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKY(Y115E) phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs.
authors.affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Yamada
authors.affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan.
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Sato
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis* / Q000737:chemistry; D044182:Benzylisoquinolines / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D039323:Coptis / Q000166:cytology / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D047468:Immunoprecipitation; D007976:Leupeptins / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D059828:Plant Cells / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D059748:Proteolysis / Q000187:drug effects; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000470:Alkaloids; D044182:Benzylisoquinolines; D007976:Leupeptins; D010940:Plant Proteins; D011480:Protease Inhibitors; D014157:Transcription Factors; D014443:Tyrosine; D004247:DNA; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde