Kozeleková Aneta, Náplavová Alexandra, Brom Tomáš, Gašparik Norbert, Šimek Jan, Houser Josef, Hritz Jozef
Central European Institute of Technology, Masaryk University, Brno, Czechia.
National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czechia.
Front Chem. 2022 Mar 7;10:835733. doi: 10.3389/fchem.2022.835733. eCollection 2022.
Protein phosphorylation is a critical mechanism that biology uses to govern cellular processes. To study the impact of phosphorylation on protein properties, a fully and specifically phosphorylated sample is required although not always achievable. Commonly, this issue is overcome by installing phosphomimicking mutations at the desired site of phosphorylation. 14-3-3 proteins are regulatory protein hubs that interact with hundreds of phosphorylated proteins and modulate their structure and activity. 14-3-3 protein function relies on its dimeric nature, which is controlled by Ser58 phosphorylation. However, incomplete Ser58 phosphorylation has obstructed the detailed study of its effect so far. In the present study, we describe the full and specific phosphorylation of 14-3-3ζ protein at Ser58 and we compare its characteristics with phosphomimicking mutants that have been used in the past (S58E/D). Our results show that in case of the 14-3-3 proteins, phosphomimicking mutations are not a sufficient replacement for phosphorylation. At physiological concentrations of 14-3-3ζ protein, the dimer-monomer equilibrium of phosphorylated protein is much more shifted towards monomers than that of the phosphomimicking mutants. The oligomeric state also influences protein properties such as thermodynamic stability and hydrophobicity. Moreover, phosphorylation changes the localization of 14-3-3ζ in HeLa and U251 human cancer cells. In summary, our study highlights that phosphomimicking mutations may not faithfully represent the effects of phosphorylation on the protein structure and function and that their use should be justified by comparing to the genuinely phosphorylated counterpart.
蛋白质磷酸化是生物学用于调控细胞过程的关键机制。为了研究磷酸化对蛋白质特性的影响，需要一个完全且特异性磷酸化的样本，尽管这并非总能实现。通常，这个问题通过在所需的磷酸化位点引入模拟磷酸化的突变来解决。14-3-3蛋白是调控蛋白枢纽，可与数百种磷酸化蛋白相互作用并调节其结构和活性。14-3-3蛋白的功能依赖于其二聚体性质，该性质受Ser58磷酸化控制。然而，迄今为止，Ser58的不完全磷酸化阻碍了对其作用的详细研究。在本研究中，我们描述了14-3-3ζ蛋白在Ser58处的完全且特异性磷酸化，并将其特性与过去使用的模拟磷酸化突变体（S58E/D）进行了比较。我们的结果表明，对于14-3-3蛋白而言，模拟磷酸化的突变不足以替代磷酸化。在14-3-3ζ蛋白的生理浓度下，磷酸化蛋白的二聚体-单体平衡比模拟磷酸化突变体的平衡更倾向于单体。寡聚状态也会影响蛋白质特性，如热力学稳定性和疏水性。此外，磷酸化改变了14-3-3ζ在HeLa和U251人癌细胞中的定位。总之，我们的研究强调，模拟磷酸化的突变可能无法忠实地代表磷酸化对蛋白质结构和功能的影响，其使用应通过与真正磷酸化的对应物进行比较来证明其合理性。
