Regulation of human methylenetetrahydrofolate reductase by phosphorylation

Abstract

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, the methyl donor for the conversion of homocysteine to methionine. Regulation of MTHFR activity is crucial for maintaining cellular concentrations of methionine and S-adenosylmethionine (AdoMet). Purified recombinant human MTHFR expressed in insect cells is multiply phosphorylated on an N-terminal extension of the protein that contains a highly conserved serine-rich region. Treatment by alkaline phosphatase removes seven phosphoryl groups from the enzyme. Thr-34 was identified as one of the seven phosphorylation sites by using a monoclonal antibody directed toward p Thr-Pro. Mutation of Thr-34 to Ala completely blocks modification as judged by mass spectrometric analysis, suggesting that Thr-34 is the priming phosphorylation site. The Thr34Ala mutant was expressed in baculovirus-infected insect cells, and its enzymic properties were compared with wild-type enzyme. The mutant enzyme and alkaline phosphatase-treated wild-type enzyme are more active than untreated wild-type enzyme and less sensitive to inhibition by saturating AdoMet, indicating that phosphorylation at Thr-34 is critical for allosteric regulation of human MTHFR activity by AdoMet. The absence of methionine and the presence of adenosine in the cell culture medium, which lead to a low intracellular AdoMet͞S-adenosylhomocysteine ratio, are associated with faster electrophoretic mobility of MTHFR, presumably because of less or no phosphorylation. Because the faster-mobility MTHFR is associated with the more active form of MTHFR, this response is likely to increase methionine production. Those observations suggest that AdoMet functions not only as an allosteric inhibitor but also to control phosphorylation of human MTHFR.homocysteine ͉ posttranslational modification

Endoplasmic Reticulum Stress Increases the Expression of Methylenetetrahydrofolate Reductase through the IRE1 Transducer

“…3D). This unidentified band is likely to be a degradation product of MTHFR, because a band of similar size was observed in Yamada et al (13) and, in our studies, this protein appeared to increase in intensity under circumstances that increased the amount of the 70-kDa MTHFR protein (as shown in Fig. 3A).…”

“…3B). As expected, we obtained complex Western blot patterns for MTHFR because there are two possible protein isoforms (with apparent mass of 70 or 77 kDa (15)) and the shorter MTHFR isoform has been shown to undergo phosphorylation (13). The stress inducers clearly increased the amount of GRP78 as well as the intensity of the MTHFR protein (short isoform of 70 kDa, phosphorylated (SP) and non-phosphorylated (S) forms).…”

“…Studies on MTHFR regulation are few in number. S-Adenosylmethionine is an allosteric regulator of MTHFR and the post-translational modification of MTHFR by phosphorylation leads to decreased activity and increased sensitivity of the enzyme to S-adenosylmethionine (13). Phosphorylation of MTHFR has been demonstrated for the smaller MTHFR isoform (70 kDa); phosphorylation of the larger isoform (77 kDa) has not been examined (13).…”

“…S-Adenosylmethionine is an allosteric regulator of MTHFR and the post-translational modification of MTHFR by phosphorylation leads to decreased activity and increased sensitivity of the enzyme to S-adenosylmethionine (13). Phosphorylation of MTHFR has been demonstrated for the smaller MTHFR isoform (70 kDa); phosphorylation of the larger isoform (77 kDa) has not been examined (13). We recently characterized 2 promoters for MTHFR, each of which may direct the synthesis of one of the two isoforms through different transcriptional start site clusters and alternative splicing (14).…”

Génétique moléculaire deMTHFR

“…Les travaux doivent se poursuivre afin de mieux identifier les régions régulatrices du gène et de comprendre leur modulation [7], ainsi que les facteurs qui influencent l'épissage alternatif de ses ARNm et la synthèse de ses deux isoformes protéiques [6]. Enfin, la régulation de l'enzyme par phosphorylation constitue un nouveau domaine à éclaircir [30]. La prochaine décennie permettra probablement d'éva-luer les répercussions cliniques du polymorphisme sur d'autres maladies.…”