Redox regulation of proteins via cysteine residue oxidation is involved in the control of various cellular signal pathways. Pyruvate kinase M2 (PKM2), a rate-limiting enzyme in glycolysis, is critical for the metabolic shift from glycolysis to the pentose phosphate pathway under oxidative stress in cancer cell growth. The PKM2 tetramer is required for optimal pyruvate kinase (PK) activity, whereas the inhibition of inter-subunit interaction of PKM2 induced by Cys358 oxidation has reduced PK activity. In the present study, we identified three oxidation-sensitive cysteine residues (Cys358, Cys423 and Cys424) responsible for four oxidation forms via the thiol oxidant diamide and/or hydrogen peroxide (H2O2). Possibly due to obstruction of the dimer-dimer interface, H2O2-induced sulfenylation (-SOH) and diamide-induced modification at Cys424 inhibited tetramer formation and PK activity. Cys423 is responsible for intermolecular disulfide bonds with heterologous proteins via diamide. Additionally, intramolecular polysulphide linkage (–Sn–, n ≧ 3) between Cys358 and an unidentified PKM2 Cys could be induced by diamide. We observed that cells expressing the oxidation-resistant PKM2 (PKM2C358,424A) produced more intracellular reactive oxygen species (ROS) and exhibited greater sensitivity to ROS-generating reagents and ROS-inducible anti-cancer drugs compared with cells expressing wild-type PKM2. These results highlight the possibility that PKM2 inhibition via Cys358 and Cys424 oxidation contributes to eliminating excess ROS and oxidative stress.
Comprehensive analyses of the cysteine thiol oxidation of PKM2 reveal the effects of multiple oxidation on cellular oxidative stress response
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Hayato Irokawa, Satoshi Numasaki, Shin Kato, Kenta Iwai, Atsushi Inose-Maruyama, Takumi Ohdate, Gi-Wook Hwang, Takashi Toyama, Toshihiko Watanabe, Shusuke Kuge; Comprehensive analyses of the cysteine thiol oxidation of PKM2 reveal the effects of multiple oxidation on cellular oxidative stress response. Biochem J 16 April 2021; 478 (7): 1453–1470. doi: https://doi.org/10.1042/BCJ20200897
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