To sense and defend against oxidative stress, cells depend on signal transduction cascades involving redox‐sensitive proteins. We previously identified SUMO (small ubiquitin‐related modifier) enzymes as downstream effectors of reactive oxygen species (ROS). Hydrogen peroxide transiently inactivates SUMO E1 and E2 enzymes by inducing a disulfide bond between their catalytic cysteines. How important their oxidation is in light of many other redox‐regulated proteins has however been unclear. To selectively disrupt this redox switch, we identified a catalytically fully active SUMO E2 enzyme variant (Ubc9 D100A) with strongly reduced propensity to maintain a disulfide with the E1 enzyme in vitro and in cells. Replacement of Ubc9 by this variant impairs cell survival both under acute and mild chronic oxidative stresses. Intriguingly, Ubc9 D100A cells fail to maintain activity of the ATM–Chk2 DNA damage response pathway that is induced by hydrogen peroxide. In line with this, these cells are also more sensitive to the ROS‐producing chemotherapeutic drugs etoposide/Vp16 and Ara‐C. These findings reveal that SUMO E1~E2 oxidation is an essential redox switch in oxidative stress.
E1 and E2 enzymes in the SUMO conjugation pathway become reversibly disulfide‐linked between their active site cysteines upon oxidative stress. A Ubc9 mutant selectively deficient for this mechanism allows to study the cellular roles of redox‐dependent global SUMOylation shutdown.
Redox regulation of SUMO E1 and E2 enzymes is conserved in different mammalian cell lines.
Ubc9 D100A is catalytically fully active but shows strongly reduced propensity to maintain a disulfide with the E1 enzyme in vitro and in cells.
Cells expressing only Ubc9 D100A have survival defects under mild oxidative stress, hydrogen peroxide and ROS‐producing chemotherapeutics.
Ubc9 D100A cells fail to maintain ROS‐induced activation of the ATM–Chk2 DNA damage response pathway.
Persistent ATM activity may require deSUMOylation of certain protein(s) as SUMO E1∼E2 crosslinking acts downstream of initial ATM phosphorylation.
The EMBO Journal (2016) 35: 1312–1329
- Received October 30, 2015.
- Revision received March 26, 2016.
- Accepted April 18, 2016.
- © 2016 The Authors
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