Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown. Here, we study multi-site phosphorylation using the yeast trehalase Nth1 and its activator, the 14-3-3 protein Bmh1, as a model. Nth1 is known to be phosphorylated by the metabolic kinase PKA on four serine residues and by the cell cycle kinase CDK on one residue. However, how these five phospho-sites adjust Nth1 activity remains unclear. Using a novel reporter construct, we investigated the contribution of the individual sites for the regulation of the trehalase and its 14-3-3 interactor. In contrast to the constitutively phosphorylated S20 and S83, the weaker sites S21 and S60 are only phosphorylated by increased PKA activity. For binding Bmh1, S83 functions as the high-affinity “gatekeeper” site, but successful binding of the Bmh1 dimer and thus Nth1 activation requires S60 as a secondary site. Under nutrient-poor conditions with low PKA activity, S60 is not efficiently phosphorylated and the cell cycle dependent phosphorylation of S66 by Cdk1 contributes to Nth1 activity, likely by providing an alternative Bmh1 binding site. Additionally, the PKA sites S20 and S21 modulate the dephosphorylation of Nth1 on downstream Bmh1 sites. In summary, our results expand our molecular understanding of Nth1 regulation and provide a new aspect of the interaction of 14-3-3 proteins with their targets.

中文翻译：

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通过多位磷酸化和14-3-3相互作用调节海藻糖酶活性

蛋白质磷酸化可以快速调节细胞活性，以适应各种细胞内和环境刺激。许多磷蛋白靶向一个以上的位点，从而可以整合多个信号并实现复杂的反应。然而，多个磷酸位点之间的层次和相互作用通常是未知的。在这里，我们研究使用酵母海藻糖酶Nth1及其激活剂14-3-3蛋白Bmh1作为模型的多位磷酸化。已知Nth1在四个丝氨酸残基上被代谢激酶PKA磷酸化，在一个残基上被细胞周期激酶CDK磷酸化。但是，这五个磷酸位点如何调节Nth1活性仍不清楚。我们使用新型的报告基因构建体，研究了海藻糖酶及其14-3-3相互作用因子调控中各个位点的贡献。与组成型磷酸化的S20和S83相反，较弱的位点S21和S60仅通过增加的PKA活性进行磷酸化。为了结合Bmh1，S83充当高亲和力的“关守”位点，但是Bmh1二聚体的成功结合以及Nth1的成功结合需要S60作为第二位点。在低PKA活性的营养贫乏条件下，S60不能有效地被磷酸化，并且Cdk1对细胞周期的依赖使S66的磷酸化有助于Nth1活性，可能是通过提供一个替代的Bmh1结合位点。此外，PKA位点S20和S21调节下游Bmh1位点上Nth1的去磷酸化。总之，我们的结果扩大了我们对Nth1调控的分子理解，并提供了14-3-3蛋白与其靶标相互作用的新方面。