The Complex Effects of PKM2 and PKM2:IP₃R Disruption on Intracellular Ca²⁺ Handling and Cellular Functions

Pyruvate kinase M (PKM) 2 was described to interact with the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) and suppress its activity. To further investigate the physiological importance of the PKM2:IP₃R interaction, we developed and characterized HeLa PKM2 knockout (KO) cells. In the HeLa PKM2 KO cells, the release of Ca²⁺ to the cytosol appears to be more sensitive to low agonist concentrations than in HeLa wild-type (WT) cells. However, upon an identical IP₃-induced Ca²⁺ release, Ca²⁺ uptake in the mitochondria is decreased in HeLa PKM2 KO cells, which may be explained by the smaller number of contact sites between the ER and the mitochondria. Furthermore, in HeLa PKM2 KO cells, mitochondria are more numerous, though they are smaller and less branched and have a hyperpolarized membrane potential. TAT-D5SD, a cell-permeable peptide representing a sequence derived from IP₃R1 that can disrupt the PKM2:IP₃R interaction, induces Ca²⁺ release into the cytosol and Ca²⁺ uptake into mitochondria in both HeLa WT and PKM2 KO cells. Moreover, TAT-D5SD induced apoptosis in HeLa WT and PKM2 KO cells but not in HeLa cells completely devoid of IP₃Rs. These results indicate that PKM2 separately regulates cytosolic and mitochondrial Ca²⁺ handling and that the cytotoxic effect of TAT-D5SD depends on IP₃R activity but not on PKM2. However, the tyrosine kinase Lck, which also interacts with the D5SD sequence, is expressed neither in HeLa WT nor PKM2 KO cells, and we can also exclude a role for PKM1, which is upregulated in HeLa PKM2 KO cells, indicating that the TAT-D5SD peptide has a more complex mode of action than anticipated.