Regulation of folate and methionine metabolism by multisite phosphorylation of human methylenetetrahydrofolate reductase.
Regulation of folate and methionine metabolism by multisite phosphorylation of human methylenetetrahydrofolate reductase.
复制标题
通过人亚甲基四氢叶酸还原酶的多位点磷酸化调节叶酸和蛋氨酸代谢。
DOI：
10.1038/s41598-019-40950-7
复制
发表时间：
2019
影响因子：
4.6
通讯作者：
Cantley,LewisC
中科院分区：
文献类型：
--
作者：
Zheng,Yuxiang;Ramsamooj,Shivan;Li,Qian;Johnson,JaredL;Yaron,TomerM;Sharra,Klaus;Cantley,LewisC
亚甲基四氢叶酸还原酶 (MTHFR) 催化 5,10-亚甲基四氢叶酸 (THF) 不可逆转化为 5-甲基-THF，从而将一碳单元投入蛋氨酸循环。虽然人们早就知道 MTHFR 会受到 S-腺苷甲硫氨酸 (SAM) 的变构抑制，但直到最近，N 端多位点磷酸化才被证明可以提供额外的调节层。在体外，MTHFR 的多重磷酸化形式对 SAM 的变构抑制更为敏感。在这里，我们试图研究负责 MTHFR 多位点磷酸化的激酶以及 MTHFR 磷酸化在细胞中的生理功能。我们在磷酸化 MTHFR 的激酶中鉴定出了 DYRK1A/2 和 GSK3A/B。此外，我们发现 MTHFR 磷酸化是通过足够的细胞 SAM 水平来维持的，这是通过 MTHFR 的 C 端 SAM 结合域来感知的。为了了解 MTHFR 磷酸化在细胞中的功能，我们生成了有效废除 MTHFR 磷酸化的 MTHFR CRISPR 敲入突变体系，并将其与亲本细胞系进行比较。尽管亲本细胞系显示出响应同型半胱氨酸处理而增加的 5-甲基-THF 产量，但敲入细胞系具有高基础水平的 5-甲基-THF 并且对同型半胱氨酸处理没有响应。总体而言，我们的结果表明 MTHFR 多位点磷酸化与 SAM 结合相协调，抑制细胞中的 MTHFR 活性。
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10-methylene-tetrahydrofolate (THF) to 5-methyl-THF, thereby committing one-carbon units to the methionine cycle. While MTHFR has long been known to be allosterically inhibited by S-adenosylmethionine (SAM), only relatively recently has N-terminal multisite phosphorylation been shown to provide an additional layer of regulation.In vitro, the multiply phosphorylated form of MTHFR is more sensitive to allosteric inhibition by SAM. Here we sought to investigate the kinases responsible for MTHFR multisite phosphorylation and the physiological function of MTHFR phosphorylation in cells. We identified DYRK1A/2 and GSK3A/B among the kinases that phosphorylate MTHFR. In addition, we found that MTHFR phosphorylation is maintained by adequate cellular SAM levels, which are sensed through the C-terminal SAM binding domain of MTHFR. To understand the function of MTHFR phosphorylation in cells, we generated MTHFR CRISPR knockin mutant lines that effectively abolished MTHFR phosphorylation and compared them with the parental cell lines. Whereas the parental cell lines showed increased 5-methyl-THF production in response to homocysteine treatment, the knockin cell lines had high basal levels of 5-methyl-THF and did not respond to homocysteine treatment. Overall, our results suggest that MTHFR multisite phosphorylation coordinates with SAM binding to inhibit MTHFR activity in cells.