Novel Function Of HECT-type Ubiquitin Ligase NEDL2in Cell Cycle Regulation

Ubiquitin-mediated proteasomal degradation represents the most critical pathwayto control the stability and quality of misfolded and specific proteins in eukaryotes.Deregulation of ubiquitin-proteasome pathway can lead to a variety of humandisorders, including neurodegenerative diseases and cancer. Protein ubiquitinationprocess is catalyzed sequentially by ubiquitin-activating enzyme E1, ubiquitinconjugating enzyme E2and ubiquitin protein ligase E3to label substrate proteins withubiquitin chains which promote their degradation in the26S proteasome. Ubiquitinligases are responsible for substrate recognition and are divided into two major classes:the really interesting new gene (RING) finger-type and homologous to E6AP carboxylterminus (HECT) domain-type ligases.The anaphase-promoting complex/cyclosome (APC/C), a RING figure-typeubiquitin ligase, is critical for controlling several transitions in the cell cycle. Theactivity of the APC/C is tightly regulated in the cell cycle. One of the main regulatorymechanisms for the APC/C is through its association with accessory-activating factors,cell division cycle protein20(Cdc20/fizzy) and fizzy/cell division cycle20related1(Cdh1/Fzr1). Cdc20associates with the APC/C from prometaphase to anaphase,whereas Cdh1recognizes mitotic cyclins and additional substrates containing either adestruction box (D box), composed of the sequence RXXL, or KEN box fordegradation in mitotic exit and G1phase.Among HECT-type ligases, there are nine members in neural precursorcell-expressed developmentally downregulated gene4(Nedd4) family. They havebeen found to be involved in many important signaling pathways, such as TGFβ, EGF,IGF, VEGF, and TNFα mediated pathways. NEDL1shares large homology withNEDL2. NEDL1is related to several cancers, including neuroblastomas, colorectalcancer and breast cancer, and it is also associated with some neurodegenerativediseases, such as amyotrophic lateral sclerosis (ALS). Compared with NEDL1andother seven members, little is known about NEDL2. NEDL2was just reported toregulate the stability of p73and is involved in the degradation of ATR kinase in laminmisexpressed cells. It will be interesting to explore its function and regulation.Using primary antibody immunoprecipitation and mass spectrometry (IP/MS),we identify a list of potential proteins that are putative NEDL2interacting proteins.The candidate list is highly enriched of mitotic proteins, especially including severalsubunits of anaphase promoting complex/cyclosome (APC/C) and Cdh1, an activatorof APC/C. Cdh1can interact with NEDL2and target it for degradation in an APC/Cdependent manner during mitotic exit. Overexpression of Cdh1reduces the level ofNEDL2, while knockdown of Cdh1increases the protein level of NEDL2. NEDL2 associates with mitotic spindles and its protein level reaches a maximum in mitosis.The function of NEDL2during mitosis is essential, as NEDL2depletion prolongs themetaphase and overexpression of NEDL2induces chromosomal lagging. NEDL2protein was highly expressed in cerebral cortex, testis, heart, kidney, and adrenal, butwas weakly detected in colon, pancreas, skin, uterus, and rectum. Next, we screenedseveral tumors of which their normal tissues expressed little NEDL2, including coloncancer, rectal cancer, cervix cancer, pancreas cancer, lung cancer, and liver cancer.The results showed that expression of NEDL2was significantly up-regulated in colonand cervix tumor tissues, compared with normal tissues. We conclude that NEDL2isa novel substrate of APC/C-Cdh1as cells exit mitosis and as a regulator of metaphaseto anaphase transition, its overexpression may contribute to tumorigenesis.In conclusion, we found NEDL2as a novel substrate of APC/C-Cdh1duringmitotic exit and the role of NEDL2in the regulation of metaphase to anaphasetransition was first revealed, and it is worthy to investigate whether NEDL2promotesthe metaphase to anaphase transition by regulating SAC inactivation or the activationof APC/C. Otherwise, overexpression NEDL2in the human tumors was firstlyuncovered, and it will be important to study the relationship between the imbalance ofNEDL2in mitotic progression and tumorigenesis.