Abstract
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.
Highlights
Oxidative modifications of protein cysteine residues represent post-translational modifications (PTMs)
A previous report indicated that a specific cysteine residue (Cys358) of Pyruvate kinase M2 (PKM2) was being involved in its redox regulation using diamide [39]
Recent global analyses on the oxidative modification of protein cysteine residues indicated that another cysteine residue (Cys424) in PKM2 was modified under oxidative stress [42,43]
Summary
Oxidative modifications of protein cysteine residues represent post-translational modifications (PTMs). Under oxidative stress conditions, increases of the metabolic flux to PPP by glycolysis inhibition serves an important role in the detoxification of H2O2 [10,11,12]. This metabolic alteration is known to be induced by the ROS-induced inactivation of glyceraldehyde-3phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) [13]. As a result, this change fulfills the metabolic requirement for cancer cells [35]. Comprehensive observation of Cys oxidation of PKM2 gives insight into the role of PKM2 in the cellular oxidative-stress response
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