Unraveling the MAX2 Protein Network in Arabidopsis thaliana: Identification of the Protein Phosphatase PAPP5 as a Novel MAX2 Interactor

The F-box protein MORE AXILLARY GROWTH 2 (MAX2) is a central component in the signaling cascade of strigolactones (SLs) as well as of the smoke derived karrikins (KARs) and the so far unknown endogenous KAI2 ligand (KL). The two groups of molecules are involved in overlapping and unique developmental processes, and signal-specific outcomes are attributed to perception by the paralogous α/β-hydrolases DWARF14 (D14) for SL and KARRIKIN INSENSITIVE 2/ HYPOSENSITIVE TO LIGHT (KAI2/HTL) for KAR/KL. Additionally, depending on which receptor is activated, specific members of the SUPPRESSOR OF MAX2 1 (SMAX1) – LIKE (SMXL) 6, 7, 8 clade control KAR/KL and SL responses respectively. As proteins that function in the same signal transduction pathway often occur in large protein complexes, we aimed at discovering new players of the MAX2, D14 and KAI2 protein network by tandem affinity purification using Arabidopsis cell cultures. When using MAX2 as a bait, various proteins were co-purified among which general components of the Skp1-Cullin-F-box complex and members of the CONSTITUTIVE PHOTOMORPHOGENIC 9 signalosome. Here, we report the identification of a novel interactor of MAX2, a type 5 serine/threonine protein phosphatase, designated PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5 (PAPP5). Quantitative affinity purification pointed at PAPP5 as being more present in KAI2 rather than D14 protein complexes. In agreement, mutant analysis suggests that PAPP5 modulates KAR/KL-dependent seed germination in suboptimal conditions and seedling development. Additionally, PAPP5 was found to dephosphorylate MAX2 in vivo independent of the synthetic strigolactone analog, rac-GR24. Together, by analyzing the protein complexes to which MAX2, D14 and KAI2 belong, we revealed a new MAX2 interactor that might act through dephosphorylating MAX2 to control mainly KAR/KL signaling.

F-box蛋白MORE AXILLARY GROWTH 2（MAX2）是独脚金内酯（strigolactones，SLs）、烟雾衍生的卡里金（karrikins，KARs）以及迄今尚未发现的内源性KAI2配体（KAI2 ligand，KL）信号级联反应的核心组分。这两类信号分子参与重叠且独特的发育调控过程，信号特异性应答由旁系同源α/β水解酶家族的不同受体介导：矮化14（DWARF14，D14）感知SL信号，而卡里金不敏感2/光不敏感（KARRIKIN INSENSITIVE 2/ HYPOSENSITIVE TO LIGHT，KAI2/HTL）则介导KAR/KL信号感知。此外，根据激活的受体类型不同，MAX2抑制因子1类似蛋白（SUPPRESSOR OF MAX2 1 LIKE，SMXL）家族中6、7、8进化支的特定成员分别调控KAR/KL与SL的信号应答。由于同一信号转导通路中的功能蛋白通常以大型蛋白复合物形式存在，本研究拟通过拟南芥细胞培养物的串联亲和纯化（tandem affinity purification）技术，挖掘MAX2、D14与KAI2蛋白互作网络中的新型调控因子。以MAX2作为诱饵蛋白进行纯化时，共获得多种共纯化蛋白，其中包括Skp1-Cullin-F-box复合体的通用组分以及组成型光形态建成9信号小体的成员。本研究鉴定得到一种新型MAX2互作蛋白：该蛋白为5型丝氨酸/苏氨酸蛋白磷酸酶，被命名为光敏色素相关蛋白磷酸酶5（PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5，PAPP5）。定量亲和纯化实验结果显示，PAPP5在KAI2复合物中的富集程度显著高于D14复合物。与之相符的是，突变体分析表明PAPP5可在亚最优条件下调控KAR/KL依赖的种子萌发与幼苗发育过程。此外，体内实验证实PAPP5能够对MAX2进行去磷酸化修饰，且该过程不依赖于合成型独脚金内酯类似物rac-GR24。综上，本研究通过解析MAX2、D14与KAI2所属的蛋白复合物组成，揭示了一种新型MAX2互作蛋白，其可能通过去磷酸化MAX2来特异性调控KAR/KL信号通路。