Meyer Cancer Center, Weill Cornell Medicine, New York, USA.
Institute for Computational Biomedicine, Department of Physiology and Biophysics, Weill Cornell Medicine, New York, USA.
Sci Rep. 2019 Mar 12;9(1):4190. doi: 10.1038/s41598-019-40950-7.
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.
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，从而将一碳单位贡献给蛋氨酸循环。虽然 MTHFR 早已被证明是受 S-腺苷甲硫氨酸（SAM）变构抑制的，但最近才发现 N 端多部位磷酸化提供了额外的调节层。在体外，MTHFR 的多磷酸化形式对 SAM 的变构抑制更敏感。在这里，我们试图研究负责 MTHFR 多部位磷酸化的激酶以及细胞中 MTHFR 磷酸化的生理功能。我们鉴定出 DYRK1A/2 和 GSK3A/B 是磷酸化 MTHFR 的激酶之一。此外，我们发现 MTHFR 磷酸化通过细胞内充足的 SAM 水平来维持，而 SAM 水平通过 MTHFR 的 C 端 SAM 结合域来感知。为了了解细胞中 MTHFR 磷酸化的功能，我们生成了有效消除 MTHFR 磷酸化的 MTHFR CRISPR 敲入突变系，并将其与亲本细胞系进行比较。虽然亲本细胞系在同型半胱氨酸处理时表现出 5-甲基-THF 产量增加，但敲入细胞系具有较高的 5-甲基-THF 基础水平，并且对同型半胱氨酸处理没有反应。总体而言，我们的结果表明，MTHFR 多部位磷酸化与 SAM 结合协调，以抑制细胞中的 MTHFR 活性。
