Phosphorylation of Arabidopsis ubiquitin ligase ATL31 is critical for plant carbon/nitrogen nutrient balance response and controls the stability of 14-3-3 proteins.
Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr(209), Ser(247), Ser(270), and Ser(303) as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr(209) and Ser(247) on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr(209) peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response.
From the Faculty of Science and Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan and.
Shigetaka
S
Yasuda
From the Faculty of Science and Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan and.
Takeo
T
Sato
From the Faculty of Science and Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan and.
Shugo
S
Maekawa
From the Faculty of Science and Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan and.
Shoki
S
Aoyama
the Plant Global Education Project, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630-0192, Japan.
Yoichiro
Y
Fukao
From the Faculty of Science and Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan and jjyama@sci.hokudai.ac.jp.
Junji
J
Yamaguchi
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D002244:Carbon / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005502:Food; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D030821:Plants, Genetically Modified / Q000201:enzymology; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology; D014026:Nicotiana / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; D002244:Carbon; C547213:ATL31 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases; D009584:Nitrogen
E3 Ubiquitin Ligase; Plasma Membrane; Protein Phosphorylation; Protein Stability; Protein-Protein Interactions; Signal Transduction