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Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination
bioRxiv - Plant Biology Pub Date : 2021-07-16 , DOI: 10.1101/2020.05.28.121228
Lukas Weiß , Lana Gaelings , Tina Reiner , Julia Mergner , Bernhard Kuster , Attila Fehér , Götz Hensel , Manfred Gahrtz , Jochen Kumlehn , Stefan Engelhardt , Ralph Hückelhoven

Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.

中文翻译:

通过磷酸化和跨界保守泛素化对植物蛋白 RACB 的 RHO 进行翻译后修饰

小的 RHO 型 G 蛋白在真核细胞中充当信号中枢和极性的主要调节器。它们的活动受到严格控制,因为有缺陷的 RHO 信号会导致异常生长和发育缺陷。两个主要过程调节 G 蛋白活性:不同核苷酸结合状态之间的规范穿梭和翻译后修饰 (PTM),后者可以支持或抑制 RHO 信号传导,具体取决于单个 PTM。在植物中,已显示植物 Rho (ROP) 信号传导活性的调节通过核苷酸交换和 GTP 水解以及脂质修饰起作用,但关于 ROP 磷酸化或泛素化的可用数据很少。因此,我们应用蛋白质组学分析来鉴定大麦 ROP RACB 的 PTM。我们观察到大麦 ROP 结合激酶 1 的体外磷酸化和 RACB 的体内泛素化。对新发现的 RACB 磷酸化位点和人类 RHO 蛋白磷酸化位点的比较分析揭示了修饰氨基酸残基的保守性,但实际磷酸化模式没有重叠。然而,鉴定的 RACB 泛素化位点在来自大麦、拟南芥和水稻的所有 ROP 以及哺乳动物 Rac1 和 Rac3 中都是保守的。该泛素化位点的点突变导致 RACB 的稳定。因此,这种高度保守的赖氨酸残基可以调节不同界的蛋白质稳定性。但实际磷酸化模式没有重叠。然而,鉴定的 RACB 泛素化位点在来自大麦、拟南芥和水稻的所有 ROP 以及哺乳动物 Rac1 和 Rac3 中都是保守的。该泛素化位点的点突变导致 RACB 的稳定。因此,这种高度保守的赖氨酸残基可以调节不同界的蛋白质稳定性。但实际磷酸化模式没有重叠。然而,鉴定的 RACB 泛素化位点在来自大麦、拟南芥和水稻的所有 ROP 以及哺乳动物 Rac1 和 Rac3 中都是保守的。该泛素化位点的点突变导致 RACB 的稳定。因此,这种高度保守的赖氨酸残基可以调节不同界的蛋白质稳定性。
更新日期:2021-07-18
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