Abstract
NEDD4-like ubiquitin ligase 2 (NEDL2) is a HECT type ubiquitin ligase. NEDL2 enhances p73 transcriptional activity and degrades ATR kinase in lamin misexpressed cells. Compared with the important functions of other HECT type ubiquitin ligase, there is less study concerning the function and regulation of NEDL2. Using primary antibody immunoprecipitation and mass spectrometry, we identify a list of potential proteins that are putative NEDL2-interacting proteins. The candidate list contains many of mitotic proteins, especially including several subunits of anaphase-promoting complex/cyclosome (APC/C) and Cdh1, an activator of APC/C. Cdh1 can interact with NEDL2 in vivo and in vitro. Cdh1 recognizes one of the NEDL2 destruction boxes (R(740)GSL(743)) and targets it for degradation in an APC/C-dependent manner during mitotic exit. Overexpression of Cdh1 reduces the protein level of NEDL2, whereas knockdown of Cdh1 increases the protein level of NEDL2 but has no effect on the NEDL2 mRNA level. NEDL2 associates with mitotic spindles, and its protein level reaches a maximum in mitosis. The function of NEDL2 during mitosis is essential because NEDL2 depletion prolongs metaphase, and overexpression of NEDL2 induces chromosomal lagging. Elevated expression of NEDL2 protein and mRNA are both found in colon cancer and cervix cancer. We conclude that NEDL2 is a novel substrate of APC/C-Cdh1 as cells exit mitosis and functions as a regulator of the metaphase to anaphase transition. Its overexpression may contribute to tumorigenesis.
Highlights
NEDD4-like ubiquitin ligase 2 (NEDL2) is a member of the HECT type ubiquitin ligase NEDD4 family, but its function remains largely unknown
We concluded that potential proteins that could interact with NEDL2 were highly enriched with mitotic proteins (Table 1)
Cdh1 maintains the activity of anaphase-promoting complex/cyclosome (APC/C) from late anaphase through G1 phase, targeting cyclin B, Cdc20, and PLK1 for degradation
Summary
NEDL2 is a member of the HECT type ubiquitin ligase NEDD4 family, but its function remains largely unknown. Significance: Our data provide a novel substrate of APC/C-Cdh and reveal an additional protein by which HECT type ubiquitin ligase can regulate mitosis. Except for NEDD4-like ubiquitin ligase 1 (NEDL1) and NEDD4-like ubiquitin ligase 2 (NEDL2), known as HECW1 and HECW2, the other seven members can regulate ubiquitin-mediated intracellular trafficking, proteasomal degradation, and nuclear translocation of multiple proteins. We first revealed that NEDL2 was localized on mitotic spindles and that its protein level reached a maximum in mitosis and was degraded by APC/C-Cdh in a D-box dependent manner during mitotic exit. NEDL2 depletion caused a marked delay of the metaphaseanaphase transition, and the overexpression of wild type or mutant NEDL2 containing the C2 domain caused earlier activation of APC/C, leading to chromosome lagging, which may induce tumorigenesis
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