DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation

Nucleic Acids Research, May 2015

The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.

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DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation

Nucleic Acids Research DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation Juan Yi 1 2 Guang Lu 2 Li Li 2 Xiaozhen Wang 0 Li Cao 2 Ming Lin 2 Sha Zhang 2 Genze Shao 1 2 0 Department of Breast Surgery, the First Hospital of Jilin University , Changchun 130021 , China 1 Institute of Systems Biology, Peking University , Beijing 100191 , China 2 Department of Cell Biology, School of Basic Medical Sciences, Peking University , Beijing 100191 , China The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damageinduced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4BRING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase. - The human genome is frequently challenged by many types of DNA damage resulting from both exogenous (environmental factors such as ultraviolet [UV]/ionizing radiation [IR]) and endogenous (cellular metabolic processes) sources (1). To counteract these threats, cells have evolved an intrinsic mechanismthe DNA damage response (DDR)to ensure genome integrity (2). In response to DNA damage, the cell cycle checkpoint is activated, leading to cell cycle arrest. Meanwhile, DNA damage repair pathways are initiated. In addition, DNA damage also induces apoptosis to remove any cells with unrepaired DNA lesions. These processes are interwoven and highly coordinated to maintain genome integrity. During the DDR process, many key proteins involved are rapidly degraded or turned over through the ubiquitinproteasome pathway. Numerous E3 ligases have been reported to play a critical role in regulating DDR, possibly through the targeting of essential factors that control these processes, leading to ubiquitination (26). The canonical protein ubiquitination process requires the participation of a series of enzymes, including E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) (7). E3 ligases are crucial in determining the substrate specificity during protein ubiqutination, thereby playing significant roles in the modulation of DDR. HUWE1 (also known as Mule, ARF-BP1, E3Histone, UREB1, HECTH9 and LASU1) is a HECT (homology to E6-AP C terminus) E3 ubiquitin ligase that is involved in diverse cellular processes, including apoptosis (8), DNA replication, DNA damage repair (913) and transcriptional regulation (1418). Among those reported functions, apoptosis and DNA damage repair seem to be the major functions that are mediated by HUWE1. Increasing evidence shows that HUWE1 is a pivotal regulator of DNA damage-induced apoptosis. The most important substrate of HUWE1 identified so far is MCL-1, a BCL-2 family member that is essential to the inhibition of apoptosis. It has been demonstrated that HUWE1-mediated ubiquitination and proteasomal degradation of MCL-1 are required to induce apoptosis in response to DNA damage (8). Another HUWE1 substrate involved in apoptosis is mitofusin 2 (Mfn2), an essential component of the fusion apparatus of the mitochondrial outer membrane. Under conditions of stress, HUWE1 targets Mfn2 for ubiquitination and proteasomal degradation, resulting in mitochondrial fragmentation, thereby leading to enhanced apoptotic cell death (19). Another apoptosis-related protein, RASSF1C, was also identified as a HUWE1 substrate (20). In addition, p53, a key effector in stress-induced apoptosis, was found to be regulated by HUWE1 through the ubiquitin-proteasomal system (UPS) (18). Previous studies have shown that HUWE1 is essential for DNA damageinduced p53 activation and apoptosis induced by histone deacetylase (HDAC) inhibitors (21). Recently, we discovered that HUWE1 mediates BRCA1 ubiquitination and degradation (13). Collectively, these findings highlight a critical role of HUWE1 in the mediation of stress-induced apoptosis and DNA damage repai (...truncated)


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Juan Yi, Guang Lu, Li Li, Xiaozhen Wang, Li Cao, Ming Lin, Sha Zhang, Genze Shao. DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation, Nucleic Acids Research, 2015, pp. 4579-4590, 43/9, DOI: 10.1093/nar/gkv325