Abstract LB-081: Elucidating the function of MCM3 ubiquitination by KEAP1: crosstalk between redox-sensing and cell cycle progression
Authors: Mulvaney Kathleen M. , Matson Jacob , Yan Feng , Goldfarb Dennis , Cook Jeannette , Major Michael Benjamin 
Published: August 2015

While the KEAP1-NRF2 axis is essential for maintaining redox homeostasis, whether KEAP1 has alternative functions and how this pathway crosstalks with other important cellular processes remains unknown. KEAP1 targets the NRF2 transcription factor for proteasomal degradation in a redox-sensitive manner. Thus, this pathway serves as the cell's primary response to elevated reactive oxygen species. Importantly, KEAP1-NRF2 are frequently mutated in cancer, most strikingly in non-small cell lung cancer, where KEAP1 or NRF2 are mutated in 20-30% of patient tumors.

Though regulation of NRF2 has long been considered the only physiologically important role for the E3 ligase KEAP1, we have determined that KEAP1 binds the master cell cycle regulator, MCM3, a subunit of the hexameric DNA replication licensing complex, MCM2-7. Excitingly, our ubiquitination assay data establish MCM3 as a new substrate for KEAP1; however, KEAP1 intriguingly does not regulate total cellular levels of MCM3. Consistent with this, we determined that only a small pool of cellular MCM3 is bound to KEAP1, suggesting that KEAP1 may bind and ubiquitinate a highly specified pool of MCM3. To determine the function of KEAP1-dependent ubiquitination of MCM3, we recently applied a new proteomics technique to map the ubiquitinated residues within MCM3 and identify these lysines within the larger MCM2-7 helicase. This mapping and protein modeling has provided new insight into the structure-function relationship of ubiquitinated MCM3.

As MCM2-7 chromatin loading is a highly coordinated, cell cycle-dependent process, we tested whether KEAP1 loaded concurrently onto chromatin. Strikingly, we found that KEAP1 indeed loads onto chromatin during G1 and unloads in late S phase in a similar fashion as the MCM complex, further suggesting KEAP1 regulates the function of this essential cell cycle regulator on chromatin. Given the role of MCM3 in cell cycle progression, we tested whether KEAP1 was required for normal G1 to S phase progression and saw that loss of KEAP1 retards S phase DNA synthesis, which is an MCM-dependent process. Intriguingly, primary, untransformed KEAP1 knockout fibroblasts show decreased growth and aberrant cell cycle patterns consistent with a defect in the G1 to S transition. Overall, these data suggest a novel function for KEAP1 in regulating the MCM complex and cell cycle progression. We postulate that KEAP1 promotes cell cycle progression in a redox-sensitive manner through its association with MCM3 and that this presents a novel mechanism by which cells may halt cell cycle to protect DNA from damage by reactive oxygen species.

Citation Format: Kathleen M. Mulvaney, Jacob Matson, Feng Yan, Dennis Goldfarb, Jeannette Cook, Michael Benjamin Major. Elucidating the function of MCM3 ubiquitination by KEAP1: crosstalk between redox-sensing and cell cycle progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-081. doi:10.1158/1538-7445.AM2015-LB-081