J Integr Plant Biol ›› 2025, Vol. 67 ›› Issue (11): 2848-2862.DOI: 10.1111/jipb.70012

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  • 收稿日期:2025-06-13 接受日期:2025-07-13 出版日期:2025-11-01 发布日期:2025-11-08

Maintaining basal B-RAF kinase activity for abscisic acid signaling via reciprocal phosphoregulation of a single serine residue

Chen Zhu1,2,3†, Tian Sang1†, Zhen Zhang1,2,3, Yubei Wang1,2,3, Zhen Lin1, Wei Wang4, Zhaobo Lang1, Jian‐Kang Zhu1, Pengcheng Wang1*   

  1. 1. Institute of Advanced Biotechnology, Institute of Homeostatic Medicine, and School of Medicine, Southern University of Science and Technology, Shenzhen 518055, China
    2. Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China
    3. University of Chinese Academy of Sciences, Beijing 100049, China
    4. Key Laboratory of Plant Stress Biology, School of Life Sciences, Henan University, Kaifeng 475004, China

    †These authors contributed equally to this article.
    *Correspondence: Pengcheng Wang (wangpc@sustech.edu.cn)
  • Received:2025-06-13 Accepted:2025-07-13 Online:2025-11-01 Published:2025-11-08
  • Supported by:
    This work was supported by the National Natural Science Foundation of China (32471500 to P.W.); National Key Research and Development Program of China (Grant No. 2021YFA1300402 to P.W.); Shenzhen Science and Technology Program, Grant KQTD20240729102038044 to J.K.Z.,P.W., and Z.La.

Abstract: The phytohormone abscisic acid (ABA) regulates plant responses to environmental stresses, development, and immunity. Under unfavorable conditions, ABA forms a complex with its receptor proteins Pyrabactin Resistance 1 (PYR1)/PYR1-likes (PYLs)/Regulatory Component of ABA Receptors (RCARs), inhibiting Clade A Protein Phosphatases Type 2C (PP2Cs) and releasing Sucrose Non-Fermenting-1-Related Protein Kinase 2s (SnRK2s) from PP2C-mediated inhibition. Rapidly Accelerated Fibrosarcoma (RAF) kinases from the B1, B2, and B3 subgroups phosphorylate and reactivate SnRK2s, initiating ABA responses. While ABA does not significantly activate B-RAFs, their basal activity is essential for initiating ABA signaling. However, the mechanisms sustaining this basal B-RAF activity are not fully understood. In this study, we revealed that Clade A PP2Cs interact with and dephosphorylate a certain number of B3 subgroup RAFs at a conserved serine residue, corresponding to Ser619 in RAF3, within the phosphate-binding loop. A phosphomimicking mutation at this residue, RAF3S619D, failed to bind ATP and exhibited diminished kinase activity in vitro and in vivo. Ser619 in RAF3 is an autophosphorylation site, phosphorylated by recombinant RAF3-KD but not by its substrate SnRK2.6. The RAF3S619A mutant, abolishing Ser619 autophosphorylation, displayed increased kinase activity in vitro. The B-RAF high-order mutant OK100-B3 carrying RAF3S619A showed enhanced ABA sensitivity compared with those with wild-type RAF3. Thus, PP2C-mediated dephosphorylation and the autophosphorylation of this unique serine residue dynamically regulate ATP binding affinity and tightly control RAF3 activity during various ABA signaling phases. This intricate mechanism ensures rapid RAF–SnRK2 cascade activation during stress while promptly desensitizing RAFs once stress signaling commences.

Key words: abiotic stress, abscisic acid (ABA), Arabidopsis, protein phosphatase 2C (PP2C), protein phosphorylation, Raf kinase

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