TY - JOUR
T1 - Comprehensive analyses of the cysteine thiol oxidation of PKM2 reveal the effects of multiple oxidation on cellular oxidative stress response
AU - Irokawa, Hayato
AU - Numasaki, Satoshi
AU - Kato, Shin
AU - Iwai, Kenta
AU - Inose-Maruyama, Atsushi
AU - Ohdate, Takumi
AU - Hwang, Giwook
AU - Toyama, Takashi
AU - Watanabe, Toshihiko
AU - Kuge, Shusuke
N1 - Funding Information:
This work was primarily supported by JSPS KAKENHI Grant Numbers 17K15030 (to H.I.), 18K06630 (to S.K.) and 21200069 (to S.K.), with additional funding from the Science Research Promotion Fund from ‘Promotion and Mutual Aid Corporation for Private Schools of Japan’ (to S.K.). TO was funded by Special Researcher Incentives (11J10274).
Publisher Copyright:
© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
PY - 2021/4
Y1 - 2021/4
N2 - 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.
AB - 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.
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U2 - 10.1042/BCJ20200897
DO - 10.1042/BCJ20200897
M3 - Article
C2 - 33749780
AN - SCOPUS:85104852644
VL - 478
SP - 1453
EP - 1470
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 7
ER -