Zhu Chen, Sang Tian, Zhang Zhen, Wang Yubei, Lin Zhen, Wang Wei, Lang Zhaobo, Zhu Jian-Kang, Wang Pengcheng
Institute of Advanced Biotechnology, Institute of Homeostatic Medicine, and School of Medicine, Southern University of Science and Technology, Shenzhen, 518055, China.
Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
J Integr Plant Biol. 2025 Aug 8. doi: 10.1111/jipb.70012.
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, RAF3, 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 RAF3 mutant, abolishing Ser619 autophosphorylation, displayed increased kinase activity in vitro. The B-RAF high-order mutant OK-B3 carrying RAF3 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.
植物激素脱落酸（ABA）调控植物对环境胁迫、发育及免疫的响应。在不利条件下，ABA与其受体蛋白吡唑啉酮抗性1（PYR1）/类PYR1（PYLs）/ABA受体调控成分（RCARs）形成复合物，抑制2C型A类蛋白磷酸酶（PP2Cs），并使蔗糖非发酵-1相关蛋白激酶2（SnRK2s）从PP2C介导的抑制中释放出来。B1、B2和B3亚组的快速加速纤维肉瘤（RAF）激酶使SnRK2s磷酸化并重新激活，从而启动ABA响应。虽然ABA不会显著激活B-RAFs，但其基础活性对于启动ABA信号传导至关重要。然而，维持这种基础B-RAF活性的机制尚未完全明确。在本研究中，我们发现A类PP2Cs与一定数量的B3亚组RAFs在磷酸盐结合环内一个保守的丝氨酸残基（对应于RAF3中的Ser619）相互作用并使其去磷酸化。该残基处的磷酸模拟突变体RAF3无法结合ATP，在体外和体内均表现出激酶活性降低。RAF3中的Ser619是一个自磷酸化位点，可被重组RAF3-KD磷酸化，但不能被其底物SnRK2.6磷酸化。消除Ser619自磷酸化的RAF3突变体在体外表现出增加的激酶活性。携带RAF3的B-RAF高阶突变体OK-B3与野生型RAF3相比，表现出增强的ABA敏感性。因此，PP2C介导的去磷酸化以及这个独特丝氨酸残基的自磷酸化在不同的ABA信号传导阶段动态调节ATP结合亲和力，并严格控制RAF3的活性。这种复杂的机制确保了在胁迫期间RAF-SnRK2级联反应的快速激活，同时一旦胁迫信号开始，能迅速使RAFs脱敏。
