Brassinosteroids (BRs) are steroid hormones essential for plant growth and development. The BR signaling pathway has been studied in some detail, however, the functions of the BRASSINOSTEROID-SIGNALING KINASE (BSK) family proteins in the pathway have remained elusive. Through forward genetics, we identified five semi-dominant mutations in the BSK3 gene causing BSK3 loss-of-function and decreased BR responses. We therefore investigated the function of BSK3, a receptor-like cytoplasmic kinase, in BR signaling and plant growth and development. We find that BSK3 is anchored to the plasma membrane via N-myristoylation, which is required for its function in BR signaling. The N-terminal kinase domain is crucial for BSK3 function, and the C-terminal three tandem TPR motifs contribute to BSK3/BSK3 homodimer and BSK3/BSK1 heterodimer formation. Interestingly, the effects of BSK3 on BR responses are dose-dependent, depending on its protein levels. Our genetic studies indicate that kinase dead BSK3 K86R protein partially rescues the bsk3-1 mutant phenotypes. BSK3 directly interacts with the BSK family proteins (BSK3 and BSK1), BRI1 receptor kinase, BSU1 phosphatase, and BIN2 kinase. BIN2 phosphorylation of BSK3 enhances BSK3/BSK3 homodimer and BSK3/BSK1 heterodimer formation, BSK3/BRI1 interaction, and BSK3/BSU1 interaction. Furthermore, we find that BSK3 upregulates BSU1 transcript and protein levels to activate BR signaling. BSK3 is broadly expressed and plays an important role in BR-mediated root growth, shoot growth, and organ separation. Together, our findings suggest that BSK3 may function as a scaffold protein to regulate BR signaling. The results of our studies provide new insights into early BR signaling mechanisms.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health (https://www.nih.gov, GM122604 to JC and GM067203 to WMG), Howard Hughes Medical Institute (http://www.hhmi.org, to JC), Human Frontier Science Program (http://www.hfsp.org) and The Pioneer Postdoctoral Endowment Fund (to BCW), European Molecular Biology Organization (http://www.embo.org, ALTF 675-2007 to YJ), and F.M. Kirby Foundation (http://fmkirbyfoundation.org) and Marc and Eva Stern Foundation (to YJ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Jack Bolado for ordering reagents, Youssef Belkhadir and Michael Hothorn for helping us identify the lysine residue to create BSK3 K86R kinase dead mutation, Kazumasa Nito for the Gateway destination vectors pTNT (GST) and pTNT (FLAG), Yanhai Yin at Iowa State University for the anti-BES1 antibody, and Roger Tsien at UCSD for the mCitrine clone. We also thank the members of the Chory laboratory for helpful discussions and Neil E. Olszewski for critical reading and comments on the manuscript.
© 2019 Ren et al.
Copyright 2019 Elsevier B.V., All rights reserved.