RETINOBLASTOMA-RELATED (RBR) proteins orchestrate cell division, differentiation, and survival in response to environmental and developmental cues through protein-protein interactions that are governed by multisite phosphorylation. Here we explore, using a large collection of transgenic RBR phosphovariants to complement protein function in Arabidopsis thaliana, whether differences in the number and position of RBR phosphorylation events cause a diversification of the protein's function. While the number of point mutations influence phenotypic strength, phosphosites contribute differentially to distinct phenotypes. RBR pocket domain mutations associate primarily with cell proliferation, while mutations in the C-region are linked to stem cell maintenance. Both phospho-mimetic and a phospho-defective variants promote cell death, suggesting that distinct mechanisms can lead to similar cell fates. We observed combinatorial effects between phosphorylated T406 and phosphosites in different protein domains, suggesting that specific, additive, and combinatorial phosphorylation events fine-tune RBR function. Suppression of dominant phospho-defective RBR phenotypes with a mutation that inhibits RBR interacting with LXCXE motifs, and an exhaustive protein-protein interaction assay, not only revealed the importance of DREAM complex members in phosphorylation-regulated RBR function but also pointed to phosphorylation-independent RBR roles in environmental responses. Thus, combinatorial phosphorylation defined and separated developmental, but not environmental, functions of RBR.

中文翻译：

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发育线索由拟南芥视网膜母细胞瘤相关蛋白 RBR1 的组合磷酸化编码。


视网膜母细胞瘤相关 （RBR） 蛋白通过受多位点磷酸化控制的蛋白质-蛋白质相互作用，响应环境和发育线索协调细胞分裂、分化和存活。在这里，我们探讨了使用大量转基因 RBR 磷酸化变体来补充拟南芥中的蛋白质功能，RBR 磷酸化事件的数量和位置的差异是否会导致蛋白质功能的多样化。虽然点突变的数量会影响表型强度，但磷酸化位点对不同表型的贡献不同。RBR 口袋结构域突变主要与细胞增殖有关，而 C 区突变与干细胞维持有关。磷酸化模拟型和磷酸化缺陷型变体都促进细胞死亡，表明不同的机制可导致相似的细胞命运。我们观察到磷酸化 T406 和磷酸化位点在不同蛋白质结构域之间的组合效应，表明特异性、加性和组合磷酸化事件微调了 RBR 功能。通过抑制 RBR 与 LXCXE 基序相互作用的突变抑制显性磷酸化缺陷 RBR 表型，以及详尽的蛋白质-蛋白质相互作用测定，不仅揭示了 DREAM 复合物成员在磷酸化调节的 RBR 功能中的重要性，还指出了磷酸化非依赖性 RBR 在环境反应中的作用。因此，组合磷酸化定义并分离了 RBR 的发育功能，而不是环境功能。