In the present study, we reveal how nuclear DNA damage signals are conveyed to the cytosolic IKK and p38 MAPK/MK2 kinases to mediate cell survival. We demonstrate crucial cytosolic roles for RIP1 and TAK1 in the NF-κB response, demonstrating that SUMO-1- and ubiquitin-modified RIP1 triggers recruitment of the ubiquitin-activated kinase TAK1. These findings indicate that in addition to its nuclear role(s), RIP1 has cytosolic functions and contributes downstream of the nuclear ATM/NEMO/RIP1 complex. Our studies also reveal a requirement for TAK1 in the NF-κB and the alternative p38 MAPK/MK2 survival pathway that is active in p53-deficient malignancies. We demonstrate that DNA damage stimulates TAK1 kinase activity and, importantly, that TAK1 kinase inhibition impairs both NF-κB and p38 MAP/MK2 kinase activation and thereby sensitizes p53-deficient human tumor cells to DNA-damaging agents. These findings have implications for translational cancer research and lead to the prediction that inhibition of TAK1 and/or RIP1 modification may sensitize p53-deficient, chemoresistant tumors to DNA-damaging agents.
Our coimmunoprecipitation studies reveal that DNA damage stimulates interactions between ATM, NEMO, RIP1, and TAK1 in the cytosol of etoposide-treated cells. These findings led us to interrogate the nature of the cytosolic complexes formed in response to DNA damage. Size exclusion chromatography revealed that DNA damage causes shifts in RIP1, NEMO, TAK1, and IKKβ proteins into overlapping fractions corresponding to a molecular-mass range of 500 to 700 kDa. Coimmunoprecipitation of column fractions confirmed that DNA damage induces the formation of a NEMO, RIP1, and TAK1 complex. Interestingly, these studies also reveal that the NEMO/TAK1 complex contains modified RIP1. Surprisingly, only a fraction of the total RIP1 protein appears to be modified and shifts into the 500- to 700-kDa complex in cells with DNA damage (A and data not shown). This is in contrast to the amount of polyubiquitinated RIP1 detected when the same cells are treated with TNF-α for 10 min (C). The reasons for these differences are unclear but may reflect the nature of the NF-κB responses. Although the TNF-induced NF-κB response is rapid and robust, occurring within minutes of receptor activation, DNA-damaging agents induce a weaker, more sustained NF-κB response, detected at 30 min and lasting several hours. The delay observed in the damage response may reflect the time required to relay the nuclear damage signal to the cytosol, whereas the duration of the DNA damage-induced NF-κB response may reflect how the pathway is negatively regulated. Cytokine-induced NF-κB responses are terminated in part by the rapid deubiquitination of cytosolic components, such as RIP1, TRAF6, and NEMO (1
). In contrast, the DNA damage-induced NF-κB pathway may be regulated in the nucleus potentially at the level of the SUMO proteases that control NEMO and/or RIP1 sumoylation.
In addition to RIP1 and NEMO, the p53-induced protein with death domain (PIDD) was also shown to interact with RIP1 to facilitate NEMO sumoylation (11
). Yet in contrast to a Rip1, Nemo, or Tak1 deficiency, Pidd-deficient fibroblasts appear to respond normally to DNA damage (17
). These findings indicate that either PIDD is not absolutely required for this NF-κB response or that redundant pathways exist in certain cell types. Consistent with this idea, the requirement for PARylation in DNA damage-induced NF-κB activation appears to be cell type dependent (22
). In contrast to the study of Janssens et al., which identified PIDD, RIP1, and NEMO in the nucleus of HEK293 cells with damaged DNA (11
), Stilmann et al. failed to detect PIDD or RIP1 as components of the nuclear PARP-1 complex that is induced in mouse embryonic fibroblasts following gamma irradiation (22
). These seemingly disparate findings could indicate that the composition of the DNA damage-induced nuclear complexes vary among cell types or that the PIDD/RIP1/NEMO nuclear complex functions downstream of the PARP-1 signalsome.
Consistent with this model, the PARP-1 signalsome is detected 10 min after DNA damage, prior to the formation of the cytosolic RIP1, NEMO, and TAK1 complex which becomes detectable between 30 min and 1 h following etoposide treatment () (22
). Hence, a delay exists between PARP-1 signalsome (10 min) formation and RIP1 and NEMO sumoylation (>30 min), suggesting that sumoylation is downstream of the PARP-1 signalsome. This study detected modified RIP1 in the cytosolic complex coincident with the detection of phospho-IκBα reactivity, thereby temporally linking the posttranslational modification(s) of RIP1 to IKK activation (A and ).
In the present study, we reveal the nature of the RIP1 modifications observed in cells with DNA damage. We find that RIP1 is SUMO and ubiquitin modified and demonstrate that PIASy silencing impairs our ability to detect both sumoylated RIP1 and NEMO in etoposide-treated cells (A and B). Moreover, we detect sumoylated RIP1 and NEMO at similar time points following DNA damage (A and data not shown), suggesting that PIASy modifies both NEMO and RIP1. We provide genetic evidence that modified RIP1 is required for NF-κB activation and the survival of cells exposed to genotoxic stimuli ().
DNA damage also stimulates the ubiquitin modification of RIP1, and sumoylated RIP1 appears to be required for RIP1 ubiquitination and TAK1 recruitment in cells with DNA damage (C, D, and C). Our preliminary analysis suggests that more than one RIP1 lysine may be modified in cells with DNA damage and that sumoylation may precede the ubiquitin modification of RIP1 (D). Moreover, silencing of the E2-conjugating enzyme UBC13 that is responsible for K63-linked polyubiquitination diminishes the NF-κB response and sensitizes multiple human cancer cell lines to DNA damage (data not shown). Two recent papers also implicate TAK1, UBC13, and K63-linked ubiquitination in the NF-κB response to DNA damage and identify critical roles for an ELKS-XIAP or a TRAF6-cIAP1 module in the genotoxic stress response (7
). These complexes could reflect signaling intermediates in a common pathway, since we detect ELKS in our etoposide-induced NEMO/RIP1/TAK1 complex (Y. Yang and M. A. Kelliher, unpublished data), or these findings could indicate that different cell types engage distinct modules. Collectively, these data reinforce crucial roles for K63-ubiquitin in the stabilization and assembly of the cytosolic DNA damage response complex and reveal that this NF-κB response utilizes ubiquitin-dependent signaling mechanisms to mediate cell cycle arrest and survival.
The sensitivity to DNA damage observed in mouse embryonic fibroblasts deficient for Nemo, Rip1, or Tak1 prompted us to test whether RIP1 and TAK1 contribute to the DNA damage response in human tumor cells. We demonstrate that RIP1 and/or TAK1 silencing in multiple human tumor cell lines results in an impaired NF-κB-dependent antiapoptotic response and sensitivity to DNA damage ( and data not shown). In addition to the DNA damage-induced NF-κB response, our studies reveal novel functions for TAK1 in the alternative p38 MAPK/MK2 checkpoint found to be active in human tumor cells that lack functional p53 (19
). We demonstrate that TAK1 silencing or TAK1 kinase inhibition impairs both the p38 MAPK/MK2 and JNK responses to DNA damage, interferes with cell cycle arrest, and sensitizes human tumor cells to etoposide treatment ().
The present study reveals RIP1 and TAK1 as critical mediators of the genotoxic stress response. Although we provide evidence that the kinase activity of TAK1 is required for NF-κB and p38 MAPK/MK2 activation, the kinase activity of RIP1 has been shown to be dispensable for the initial NF-κB response to DNA damage (10
). Our data establish a requirement for modified RIP1 in the NF-κB response to DNA damage, raising the possibility that the SUMO-1 or ubiquitin modifying enzymes that regulate the genotoxic stress response could serve as new therapeutic targets in chemoresistance. Based on the results of our studies, these targets would be predicted to inhibit the NF-κB response and sensitize resistant tumor cells to DNA-damaging agents without the immune-associated side effects associated with complete IKK inhibition.