Brown N M, Anderson S A, Steffen D W, Carpenter T B, Kennedy M C, Walden W E, Eisenstein R S
Department of Microbiology and Immunology, University of Illinois, 835 South Wolcott Avenue, Chicago, IL 60612, USA.
Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15235-40. doi: 10.1073/pnas.95.26.15235.
Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs). IRPs modulate mRNA utilization by binding to iron-responsive elements (IRE) in the 5' or 3' untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase. The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its [4Fe-4S] cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase. IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined. To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically. However, when exposed to oxygen, the [4Fe-4S] cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1. Our findings suggest that stability of the Fe-S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and [4Fe-4S] forms of IRP1 can be modulated independently of cellular iron status. Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression.
动物主要通过铁调节蛋白（IRP）的作用来调节铁代谢。IRP通过与参与铁稳态或能量产生的蛋白质编码mRNA的5'或3'非翻译区中的铁反应元件（IRE）结合来调节mRNA的利用。IRP1也是顺乌头酸酶的胞质同工型。IRP1的活性相互排斥，并通过其[4Fe-4S]簇的组装/拆卸来调节，在IRE结合蛋白和胞质顺乌头酸酶之间可逆地转换。IRP1也受蛋白激酶C的磷酸化调节，但翻译后磷酸化增加IRE结合活性的机制尚未完全明确。为了研究这一点，将蛋白激酶C磷酸化位点Ser-138（S138）突变为模拟磷酸化的谷氨酸（S138E）、天冬氨酸（S138D）或不可磷酸化的丙氨酸（S138A）。S138E IRP1突变体以及程度较轻的S138D IRP1突变体在需氧生长的酵母中顺乌头酸酶功能受损，但在厌氧生长时未受损。纯化的野生型和突变型IRP1在厌氧条件下可重组为有活性的顺乌头酸酶。然而，当暴露于氧气时，S138D和S138E突变体的[4Fe-4S]簇分别比野生型IRP1衰变快5倍和20倍。我们的研究结果表明，IRP1的Fe-S簇的稳定性可通过磷酸化来调节，并揭示了一种机制，通过该机制IRP1的IRE结合形式和[4Fe-4S]形式之间的平衡可以独立于细胞铁状态进行调节。此外，我们的结果表明，IRP1可以作为基因表达的氧调节转录后调节因子发挥作用。
