Abstract
Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme and transcriptional coactivator and is critical for tumor metabolism. In cancer cells, native tetrameric PKM2 is phosphorylated or acetylated, which initiates a switch to a dimeric/monomeric form that translocates into the nucleus, causing oncogene transcription. However, it is not known how these post-translational modifications (PTMs) disrupt the oligomeric state of PKM2. We explored this question via crystallographic and biophysical analyses of PKM2 mutants containing residues that mimic phosphorylation and acetylation. We find that the PTMs elicit major structural reorganization of the fructose 1,6-bisphosphate (FBP), an allosteric activator, binding site, impacting the interaction with FBP and causing a disruption in oligomerization. To gain insight into how these modifications might cause unique outcomes in cancer cells, we examined the impact of increasing the intracellular pH (pHi) from ∼7.1 (in normal cells) to ∼7.5 (in cancer cells). Biochemical studies of WT PKM2 (wtPKM2) and the two mimetic variants demonstrated that the activity decreases as the pH is increased from 7.0 to 8.0, and wtPKM2 is optimally active and amenable to FBP-mediated allosteric regulation at pHi 7.5. However, the PTM mimetics exist as a mixture of tetramer and dimer, indicating that physiologically dimeric fraction is important and might be necessary for the modified PKM2 to translocate into the nucleus. Thus, our findings provide insight into how PTMs and pH regulate PKM2 and offer a broader understanding of its intricate allosteric regulation mechanism by phosphorylation or acetylation.
Highlights
Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP formation and is critical for cancer metabolism
To understand whether acetylation or phosphorylation of PKM2 influences its activity in vitro and to gain insight into how FBP influences the activities of the variants, pyruvate kinase assays were conducted at a pH of 7.5, in the absence and presence of FBP
Activity assays with the phosphoserine mimetics, PKM2 S37E and PKM2 S37D, show ;4-fold reduction in turnover numbers compared with WT PKM2 (wtPKM2) at pH 7.5 in the absence of FBP (Fig. 1A and Table S1)
Summary
Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP formation and is critical for cancer metabolism. A complex of PKM2 S37D and PIN1 appeared as a mixture of tetramer and monomer, suggesting that PIN1 mediates a change in the oligomeric state of PKM2 S37D [19] Likewise, another phosphoserine-mimicking variant, PKM2 S37E, exists in the dimeric state, as observed on a nondenaturing gel using whole-cell lysates [18]. Using whole-cell lysate of PKM2 acetylated at Lys433 and a recombinant acetyl-lysine mimetic variant of PKM2 (hereafter referred to as PKM2 K433Q), gel filtration studies determined that the enzyme appeared in the dimeric and a mixture of monomeric/dimeric states, respectively, resulting in its translocation to the nucleus [6, 8]. Understanding the mechanism of tetramer-to-dimer conversion by phosphorylation or acetylation and identification of critical residues will aid in the development of mechanism-based smallmolecule inhibitors targeted toward specific allosteric sites to stabilize the dimeric form of PKM2
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