蛋白质磷酸化是生物学用来控制细胞过程的关键机制。为了研究磷酸化对蛋白质性质的影响,一个完全和特异性磷酸化的样品是必需的,虽然并不总是可以实现。通常,这个问题可以通过在所需的磷酸化位点安装磷酸化突变来克服。14-3-3蛋白是与数百种磷酸化蛋白相互作用并调节其结构和活性的调节蛋白中心。14-3-3蛋白质功能依赖于其二聚体性质,由Ser58磷酸化控制。然而,Ser58的不完全磷酸化阻碍了迄今为止对其作用的详细研究.在本研究中,我们描述了Ser58上14-3-3ζ蛋白的完全和特异性磷酸化,并将其特征与过去使用的磷酸化突变体(S58E/D)进行了比较.我们的结果表明,在14-3-3蛋白的情况下,磷酸化突变不足以替代磷酸化。在14-3-3ζ蛋白的生理浓度下,磷酸化蛋白质的二聚体-单体平衡比磷酸化突变体更偏向单体。寡聚状态还影响蛋白质性质,例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人类癌细胞中的定位。总之,我们的研究强调,磷酸化突变可能不能忠实地代表磷酸化对蛋白质结构和功能的影响,并且应通过与真正磷酸化的对应物进行比较来证明其使用的合理性.
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.