Phosphorylation of Yeast Hexokinase 2 Regulates Its Nucleocytoplasmic Shuttling
Phosphorylation of Yeast Hexokinase 2 Regulates Its Nucleocytoplasmic Shuttling
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DOI：
10.1074/jbc.m112.401679
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发表时间：
2012-12-07
影响因子：
4.8
通讯作者：
Moreno, Fernando
中科院分区：
文献类型：
--
作者：
Fernandez-Garcia, Paula;Pelaez, Rafael;Moreno, Fernando
最近有报道葡萄糖水平诱导Hxk 2的核质穿梭。在这里，我们提出的证据表明，Hxk 2核质交通的丝氨酸14的磷酸化和去磷酸化调节。此外，我们确定了蛋白激酶Snf 1和蛋白磷酸酶Glc 7-Reg 1作为新的监管合作伙伴的核质穿梭的Hxk 2。功能研究表明，与野生型蛋白相反，Hxk 2的去磷酸化模拟突变体在低葡萄糖条件下保留其核定位，而Hxk 2的磷酸化模拟突变体在高葡萄糖条件下保留其细胞质定位。Hxk 2与Kap 60和Xpo 1的相互作用实验表明，Hxk 2的S14 D突变体的核输入严重减少，但输出显着增强。相反，Hxk 2的S14 A突变体的核输入显著增强，尽管输出严重减少。发现Hxk 2与Kap 60和Xpo 1的相互作用分别发生在蛋白质的去磷酸化和磷酸化状态。此外，我们发现Hxk 2是Snf 1的底物。突变分析表明，丝氨酸14是一个主要的在体外和体内磷酸化位点的Snf 1。我们还提供了证据，Hxk 2在丝氨酸14的去磷酸化是一个蛋白磷酸酶Glc 7-Reg 1依赖的过程。总之，这项研究建立了Hxk 2，Reg 1和Snf 1信号之间的功能联系，这涉及到Hxk 2核质穿梭的丝氨酸14的磷酸化-去磷酸化的调节。
Nucleocytoplasmic shuttling of Hxk2 induced by glucose levels has been reported recently. Here we present evidence that indicates that Hxk2 nucleocytoplasmic traffic is regulated by phosphorylation and dephosphorylation at serine 14. Moreover, we identified the protein kinase Snf1 and the protein phosphatase Glc7-Reg1 as novel regulatory partners for the nucleocytoplasmic shuttling of Hxk2. Functional studies revealed that, in contrast to the wild-type protein, the dephosphorylation-mimicking mutant of Hxk2 retains its nuclear localization in low glucose conditions, and the phosphomimetic mutant of Hxk2 retains its cytoplasmic localization in high glucose conditions. Interaction experiments of Hxk2 with Kap60 and Xpo1 indicated that nuclear import of the S14D mutant of Hxk2 is severely decreased but that the export is significantly enhanced. Conversely, nuclear import of the S14A mutant of Hxk2 was significantly enhanced, although the export was severely decreased. The interaction of Hxk2 with Kap60 and Xpo1 was found to occur in the dephosphorylated and phosphorylated states of the protein, respectively. In addition, we found that Hxk2 is a substrate for Snf1. Mutational analysis indicated that serine 14 is a major in vitro and in vivo phosphorylation site for Snf1. We also provide evidence that dephosphorylation of Hxk2 at serine 14 is a protein phosphatase Glc7-Reg1-dependent process. Taken together, this study establishes a functional link between Hxk2, Reg1, and Snf1 signaling, which involves the regulation of Hxk2 nucleocytoplasmic shuttling by phosphorylation-dephosphorylation of serine 14.