A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca²⁺ signalling through IP₃ receptors

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 ⁴⁵Ca²⁺ 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.