Polo-like kinase 1 (PLK1)-dependent phosphorylation of methylenetetrahydrofolate reductase (MTHFR) regulates replication via histone methylation

Abstract

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the folate cycle and its genetic variations have been associated with various human diseases. Previously we identified that MTHFR is phosphorylated by cyclin-dependent kinase 1 (CDK1) at T34 and MTHFR underlies heterochromatin maintenance marked by H3K9me3 levels. Herein we demonstrate that pT34 creates a binding motif that docks MTHFR to the polo-binding domain (PBD) of polo-like kinase 1 (PLK1), a fundamental kinase that orchestrates many cell cycle events. We show that PLK1 phosphorylates MTHFR at T549 in vitro and in vivo. Further, we uncovered a role of MTHFR in replication. First, MTHFR depletion increased the fraction of cells in S phase. This defect could not be rescued by siRNA resistant plasmids harboring T549A, but could be restored by overproduction of Suv4-20H2, the H4K20 methyltransferase. Moreover, siMTHFR attenuated H4K20me3 levels, which could be rescued by Suv4-20H2 overproduction. More importantly, we also investigated MTHFR-E429A, the protein product of an MTHFR single nucleotide variant. MTHFR-E429A overexpression also increased S phase cells and decreased H4K20me3 levels, and it is linked to a poor glioma prognosis in the Chinese population. Collectively, we have unveiled a vital role of PLK1-dependent phosphorylation of MTHFR in replication via histone methylation, and implicate folate metabolism with glioma.

Structural basis of human 5,10-methylenetetrahydrofolate reductase (MTHFR) regulation by phosphorylation and S-adenosylmethionine inhibition

“…Interestingly, two phosphorylated Ser are located within the FAD binding site, although their physiological significance is currently unclear. Contrary to the recent observation of Li et al 17 we did not identify phosphorylation of Thr549.…”

“…In this regard, phosphorylation allows linkage of the methionine cycle to other cellular pathways (e.g. cell cycle) through specific kinase activities (as suggested by 16,17 ).…”

“…Although phosphorylation mapping of this region has been thus far unsuccessful, scanning mutagenesis has revealed substitution of alanine for threonine at position 34 (p.Thr34Ala) to almost completely block phosphorylation 14,15 , suggesting Thr34 is the priming position. The cellular relevance of this modification remains unclear, although one group has suggested that phosphorylation at Thr34 can be accomplished by CDK1/cyclin B1 16 and at Thr549 by polo-like kinase 1 17 whereby they posit a role in histone methylation and replication.…”

O-GlcNAcylation of myosin phosphatase targeting subunit 1 (MYPT1) dictates timely disjunction of centrosomes

“…Cells were transfected with Flag-PLK1, and then treated with TMG plus glucose or left untreated. Then Flag-Plk1 was IPed from cellular extracts and incubated with GST-MTHFR (methylenetetrahydrofolate reductase), as our lab has demonstrated and MTHFR is a PLK1 substrate (37). Using the phospho-specific pThr-549 antibody, we observed an increase of kinase activity after TMG plus glucose treatment.…”

“…The IP-phosphatase assay was performed as before (22,24). The IP-kinase assay was described (37). Recombinant active Plk1 was purchased (Carna Biosciences,…”