RESUMO
The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/química , Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Citidina Trifosfato/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células HeLa , Humanos , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
Signal transduction by small ubiquitin-like modifier (SUMO) regulates a myriad of nuclear processes. Here we report on the role of SUMO in mitosis in human cell lines. Knocking down the SUMO conjugation machinery results in a delay in mitosis and defects in mitotic chromosome separation. Searching for relevant SUMOylated proteins in mitosis, we identify the anaphase-promoting complex/cyclosome (APC/C), a master regulator of metaphase to anaphase transition. The APC4 subunit is the major SUMO target in the complex, containing SUMO acceptor lysines at positions 772 and 798. SUMOylation is crucial for accurate progression of cells through mitosis and increases APC/C ubiquitylation activity toward a subset of its targets, including the newly identified target KIF18B. Combined, our findings demonstrate the importance of SUMO signal transduction for genome integrity during mitotic progression and reveal how SUMO and ubiquitin cooperate to drive mitosis.
Assuntos
Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Mitose/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/genética , Ubiquitinas/metabolismo , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/genética , Linhagem Celular Tumoral , Células HCT116 , Células HeLa , Humanos , Cinesinas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinas/genéticaRESUMO
The Anaphase Promoting Complex/Cyclosome (APC/C) is a ubiquitin E3 ligase that functions as the gatekeeper to mitotic exit. APC/C activity is controlled by an interplay of multiple pathways during mitosis, including the spindle assembly checkpoint (SAC), that are not yet fully understood. Here, we show that sumoylation of the APC4 subunit of the APC/C peaks during mitosis and is critical for timely APC/C activation and anaphase onset. We have also identified a functionally important SUMO interacting motif in the cullin-homology domain of APC2 located near the APC4 sumoylation sites and APC/C catalytic core. Our findings provide evidence of an important regulatory role for SUMO modification and binding in affecting APC/C activation and mitotic exit.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/genética , Proteínas do Citoesqueleto/genética , Mitose/genética , Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/química , Domínio Catalítico/genética , Proteínas do Citoesqueleto/química , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/genética , Plasmídeos/genética , Ligação Proteica , Conformação Proteica , Fuso Acromático/química , Fuso Acromático/genética , Sumoilação/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
MicroRNA-452 (miRNA-452) was overexpressed in docetaxel-resistant human breast cancer MCF-7 cells (MCF-7/DOC). However, its role in modulating the sensitivity of breast cancer cells to docetaxel (DOC) remains unclear. The aim of this study is to investigate the role of miRNA-452 in the sensitivity of breast cancer cells to DOC.Real-time quantitative PCR (RT-qPCR) were used to identify the differential expression of miRNA-452 between MCF-7/DOC and MCF-7 cells. MiRNA-452 mimic was transfected into MCF-7 cells and miRNA-452 inhibitor was transfected into MCF-7/DOC cells. The role of miRNA-452 in these transfected cells was evaluated using RT-qPCR, MTT assay, and flow cytometry assay. The relationship of miRNA-452 and its predictive target gene "anaphase-promoting complex 4" (APC4) was analyzed by RT-qPCR and Western blot.MiRNA-452 showed significantly higher expression (78.9-folds) in MCF-7/DOC cells compared to parental MCF-7 cells. The expression of miRNA-452 in the mimic transfected MCF-7 cells was upregulated 212.2-folds (P < 0.05) compared to its negative control (NC), and the half maximal inhibitory concentration (IC50) value of DOC (1.98 ± 0.15 µM) was significantly higher than that in its NC (0.85 ± 0.08 µM, P < 0.05) or blank control (1.01 ± 0.19 µM, P < 0.05). Furthermore, its apoptotic rate (6.3 ± 1.3 %) was distinctly decreased compared with that in its NC (23.8 ± 6.6 %, P < 0.05) or blank control (18.6 ± 4.7 %, P < 0.05). In contrast, the expression of miRNA-452 in the inhibitor-transfected MCF-7/DOC cells was downregulated 0.58-fold (P < 0.05) compared to its NC, the IC50 value of DOC (44.5 ± 3.2 µM) was significantly lower than that in its NC (107.3 ± 6.63 µM, P < 0.05) or blank control (102.22 ± 11.34 µM, P < 0.05), and the apoptotic rate (45.5 ± 10.8 %) was distinctly increased compared with its NC (9.9 ± 2.2 %, P < 0.05) and blank control (9.4 ± 2.5 %, P < 0.05). Further, there was an inverse association between miRNA-452 and APC4 expression in breast cancer cells in vitro.Dysregulation of miRNA-452 involved in the DOC resistance formation of breast cancer cells may be, in part, via targeting APC4.
