Phospho-Mutant Activity Assays Provide Evidence for the Negative Regulation of Transcriptional Regulator PRE1 by Phosphorylation
Abstract
:1. Introduction
2. Results
2.1. Phosphorylation at Ser-67 Negatively Regulated the Functions of PRE1
2.2. Mutation of the 67th Serine to Glutamic Acid Reduced the Binding Ability of PRE1 to IBH1
2.3. PRE1S67E Failed to Abolish the Interaction between IBH1 and HBI1
2.4. PRE1S67E Failed to Suppress the Dwarf Phenotypes of IBH1-Ox
2.5. Overexpression of PRE1S67E Increase the Biomass of the Plant
3. Discussion
4. Materials and Methods
4.1. Plant Materials and Growth Conditions
4.2. Vector Construction
4.3. Quantitative Real-Time PCR Analysis
4.4. Yeast Two-hybrid assays
4.5. β-gal Activity Analysis
4.6. Yeast Three-Hybrid Assays
4.7. Bimolecular Fluorescence Complementation (BiFC) Assays
4.8. Ratiometric Bimolecular Fluorescence Complementation (rBiFC) Assays
4.9. In Vitro Pull-down Assays
4.10. Coimmunoprecipitation Assays
4.11. In Vitro Kinase Assays
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
PRE | Paclobutrazol-resistance |
Ser, S | Serine |
Glu, E | glutamic acid |
Ala, A | Alanine |
BR | Brassinosteroid |
GA | Gibberellin |
ILI1 | Increased leaf inclination1 |
IBH1 | ILI1 binding bHLH |
HBI1 | Homolog of BEE 2 interacting with IBH1 |
BIN2 | GSK3-like kinase brassinosteroid-insensitive 2 |
PIFs | Phytochrome interacting factors |
GST | Glutathione S-transferase |
YFP | Yellow fluorescence protein |
MBP | Maltose-binding protein |
3-AT | 3-amino-1,2,4-triazole |
BIFC | Bimolecular fluorescence complementation |
rBiFC | Ratiometric bimolecular fluorescence complementation |
BD | Binding domain |
AD | Acting domain |
RT-PCR | Quantitative real-time PCR |
COIP | Coimmunoprecipitation |
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Wang, M.; Tian, Y.; Han, C.; Zhou, C.; Bai, M.-Y.; Fan, M. Phospho-Mutant Activity Assays Provide Evidence for the Negative Regulation of Transcriptional Regulator PRE1 by Phosphorylation. Int. J. Mol. Sci. 2020, 21, 9183. https://doi.org/10.3390/ijms21239183
Wang M, Tian Y, Han C, Zhou C, Bai M-Y, Fan M. Phospho-Mutant Activity Assays Provide Evidence for the Negative Regulation of Transcriptional Regulator PRE1 by Phosphorylation. International Journal of Molecular Sciences. 2020; 21(23):9183. https://doi.org/10.3390/ijms21239183
Chicago/Turabian StyleWang, Minmin, Yanchen Tian, Chao Han, Chuanen Zhou, Ming-Yi Bai, and Min Fan. 2020. "Phospho-Mutant Activity Assays Provide Evidence for the Negative Regulation of Transcriptional Regulator PRE1 by Phosphorylation" International Journal of Molecular Sciences 21, no. 23: 9183. https://doi.org/10.3390/ijms21239183