Nandi Suparno, Razzaghi Mortezaali, Srivastava Dhiraj, Dey Mishtu
Department of Chemistry, University of Iowa, Iowa City, Iowa, USA.
Department of Chemistry, University of Iowa, Iowa City, Iowa, USA.
J Biol Chem. 2020 Dec 18;295(51):17425-17440. doi: 10.1074/jbc.RA120.015800.
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 (pH) 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 pH 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.
丙酮酸激酶肌肉同工型2（PKM2）是一种关键的糖酵解酶和转录共激活因子，对肿瘤代谢至关重要。在癌细胞中，天然的四聚体PKM2会发生磷酸化或乙酰化，从而引发向二聚体/单体形式的转变，这种形式会转移到细胞核中，导致癌基因转录。然而，尚不清楚这些翻译后修饰（PTM）如何破坏PKM2的寡聚状态。我们通过对含有模拟磷酸化和乙酰化残基的PKM2突变体进行晶体学和生物物理分析来探究这个问题。我们发现，这些PTM引发了变构激活剂1,6-二磷酸果糖（FBP）结合位点的重大结构重组，影响了与FBP的相互作用并导致寡聚化的破坏。为了深入了解这些修饰如何在癌细胞中导致独特的结果，我们研究了将细胞内pH值从约7.1（正常细胞中）提高到约7.5（癌细胞中）的影响。对野生型PKM2（wtPKM2）和两种模拟变体的生化研究表明，随着pH值从7.0增加到8.0，活性降低，并且wtPKM2在pH 7.5时具有最佳活性并易于接受FBP介导的变构调节。然而，PTM模拟物以四聚体和二聚体的混合物形式存在，这表明生理上的二聚体部分很重要，可能是修饰后的PKM2转移到细胞核所必需的。因此，我们的研究结果深入了解了PTM和pH如何调节PKM2，并提供了对其通过磷酸化或乙酰化进行的复杂变构调节机制的更广泛理解。
