Phosphorylation Regulates myo-Inositol-3-phosphate Synthase A NOVEL REGULATORY MECHANISM OF INOSITOL BIOSYNTHESIS
Phosphorylation Regulates myo-Inositol-3-phosphate Synthase A NOVEL REGULATORY MECHANISM OF INOSITOL BIOSYNTHESIS
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DOI：
10.1074/jbc.m113.479121
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发表时间：
2013-09-13
影响因子：
4.8
通讯作者：
Greenberg, Miriam L.
中科院分区：
文献类型：
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作者：
Deranieh, Rania M.;He, Quan;Greenberg, Miriam L.
肌醇 3-磷酸合酶 (MIPS) 在肌醇稳态中发挥着至关重要的作用。编码基因 INO1 的转录受到高度调控。然而，酶的调节尚不明确。我们之前表明 MIPS 被丙戊酸间接抑制，表明该酶受到翻译后调节。使用 P-32(i) 标记和磷酸氨基酸分析，我们表明酵母 MIPS 是一种磷蛋白。质谱分析确定了五个磷酸位点，其中三个在人类 MIPS 中是保守的。对磷酸化缺陷和拟磷酸位点突变体的分析表明，酵母（Ser-184、Ser-296 和 Ser-374）和人类（Ser-177、Ser-279 和 Ser-357）中的三个保守位点影响 MIPS 活性。 S296A和S296D酵母突变体以及S177A和S177D人类突变体均表现出酶活性降低，表明丝氨酸残基在该位置至关重要。拟磷酸化突变 S184D（人 S279D）和 S374D（人 S357D）但磷酸缺陷突变不会降低活性，表明这两个位点的磷酸化具有抑制性。双突变 S184A/S374A 导致 MIPS 活性增加，赋予生长优势，并部分挽救对丙戊酸的敏感性。我们的研究结果确定了一种通过 MIPS 磷酸化调节肌醇合成的新机制。
myo-Inositol-3-phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate, suggesting that the enzyme is post-translationally regulated. Using P-32(i) labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site mutants indicated that the three conserved sites in yeast (Ser-184, Ser-296, and Ser-374) and humans (Ser-177, Ser-279, and Ser-357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phosphodeficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374A caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to valproate. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS.