A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca2+ signalling through IP3 receptors
A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca2+ signalling through IP3 receptors
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DOI：
10.1016/j.bbamcr.2021.119206
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发表时间：
2022-01-15
影响因子：
5.1
通讯作者：
Distelhorst, Clark W.
中科院分区：
文献类型：
--
作者：
Lavik, Andrew R.;McColl, Karen S.;Distelhorst, Clark W.
丙酮酸激酶亚型M2（PKM 2）是一种在胚胎组织中广泛表达的限速糖酵解酶。PKM 2在胚胎发生后的一些组织中表达下降，而其他丙酮酸激酶同工酶则上调。然而，PKM 2在癌细胞中高度表达，并且被认为在肿瘤形成期间在支持合成代谢过程中发挥作用。在这项研究中，PKM 2被确定为肌醇1，4，5-三磷酸受体（IP 3R）的相互作用蛋白质的质谱。PKM 2：IP 3R相互作用的进一步特征在于下拉和免疫共沉淀测定，这表明PKM 2与所有三种IP 3R亚型相互作用。荧光显微镜观察发现IP 3R和PKM 2均定位于内质网。PKM 2在高度保守的21个氨基酸位点（对应于小鼠1型IP 3R同种型中的氨基酸2078-2098）与IP 3R结合。基于该位点的氨基酸序列，合成肽（表示为“TAT-D5 SD”和“D5 SD”）破坏PKM 2：IP 3R相互作用，并在完整细胞（TAT-D5 SD肽）和透化细胞（D5 SD肽）的单向Ca-45（2+）通量测定中增强IP 3R介导的Ca 2+释放。TAT-D5 SD肽对PKM 2的酶活性没有影响。使用siRNA降低PKM 2蛋白表达增加了完整细胞中IP 3R介导的Ca 2+信号传导，而不改变ER Ca 2+含量。这些数据将PKM 2鉴定为抑制细胞内Ca 2+信号传导的IP 3R相互作用蛋白。因此，PKM 2在癌细胞中的表达升高不仅与其在糖酵解代谢中的典型作用有关，而且PKM 2在调节细胞内信号传导中也具有新的非典型作用。
Pyruvate kinase isoform M2 (PKM2) is a rate-limiting glycolytic enzyme that is widely expressed in embryonic tissues. The expression of PKM2 declines in some tissues following embryogenesis, while other pyruvate kinase isozymes are upregulated. However, PKM2 is highly expressed in cancer cells and is believed to play a role in supporting anabolic processes during tumour formation. In this study, PKM2 was identified as an inositol 1,4,5-trisphosphate receptor (IP3R)-interacting protein by mass spectrometry. The PKM2:IP3R interaction was further characterized by pull-down and co-immunoprecipitation assays, which showed that PKM2 interacted with all three IP3R isoforms. Moreover, fluorescence microscopy indicated that both IP3R and PKM2 localized at the endoplasmic reticulum. PKM2 binds to IP3R at a highly conserved 21-amino acid site (corresponding to amino acids 2078-2098 in mouse type 1 IP3R isoform). Synthetic peptides (denoted 'TAT-D5SD' and 'D5SD'), based on the amino acid sequence at this site, disrupted the PKM2:IP3R interaction and potentiated IP3R-mediated Ca2+ release both in intact cells (TAT-D5SD peptide) and in a unidirectional Ca-45(2+) flux assay on permeabilized cells (D5SD peptide). The TAT-D5SD peptide did not affect the enzymatic activity of PKM2. Reducing PKM2 protein expression using siRNA increased IP3R-mediated Ca2+ signalling in intact cells without altering the ER Ca2+ content. These data identify PKM2 as an IP3R-interacting protein that inhibits intracellular Ca2+ signalling. The elevated expression of PKM2 in cancer cells is therefore not solely connected to its canonical role in glycolytic metabolism, rather PKM2 also has a novel non-canonical role in regulating intracellular signalling.