The E3 ubiquitin ligase APC / C C dh1 degrades MCPH 1 after MCPH1‐βTr CP 2‐Cdc25A‐mediated mitotic entry to ensure neurogenesis
2017; Springer Nature; Volume: 36; Issue: 24 Linguagem: Inglês
10.15252/embj.201694443
ISSN1460-2075
AutoresXiaoqian Liu, Wen Zong, Tangliang Li, Yujun Wang, Xingzhi Xu, Zhong‐Wei Zhou, Zhao‐Qi Wang,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle17 November 2017free access Transparent process The E3 ubiquitin ligase APC/CCdh1 degrades MCPH1 after MCPH1-βTrCP2-Cdc25A-mediated mitotic entry to ensure neurogenesis Xiaoqian Liu Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany Search for more papers by this author Wen Zong Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany Search for more papers by this author Tangliang Li Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany Institute of Aging Research, School of Medicine, Hangzhou Normal University, Hangzhou, China Search for more papers by this author Yujun Wang Division of Biology, City of Hope National Medical Center/Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Xingzhi Xu Beijing Key Laboratory of DNA Damage Response, College of Life Sciences, Capital Normal University, Beijing, China Guangdong Key Laboratory for Genome Stability & Disease Prevention, Shenzhen University School of Medicine, Shenzhen, Guangdong, China Search for more papers by this author Zhong-Wei Zhou Corresponding Author [email protected] orcid.org/0000-0001-7971-2921 Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany Search for more papers by this author Zhao-Qi Wang Corresponding Author [email protected] orcid.org/0000-0002-8336-3485 Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany Faculty of Biology and Pharmacy, Friedrich-Schiller University of Jena, Jena, Germany Search for more papers by this author Xiaoqian Liu Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany Search for more papers by this author Wen Zong Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany Search for more papers by this author Tangliang Li Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany Institute of Aging Research, School of Medicine, Hangzhou Normal University, Hangzhou, China Search for more papers by this author Yujun Wang Division of Biology, City of Hope National Medical Center/Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Xingzhi Xu Beijing Key Laboratory of DNA Damage Response, College of Life Sciences, Capital Normal University, Beijing, China Guangdong Key Laboratory for Genome Stability & Disease Prevention, Shenzhen University School of Medicine, Shenzhen, Guangdong, China Search for more papers by this author Zhong-Wei Zhou Corresponding Author [email protected] orcid.org/0000-0001-7971-2921 Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany Search for more papers by this author Zhao-Qi Wang Corresponding Author [email protected] orcid.org/0000-0002-8336-3485 Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany Faculty of Biology and Pharmacy, Friedrich-Schiller University of Jena, Jena, Germany Search for more papers by this author Author Information Xiaoqian Liu1, Wen Zong1, Tangliang Li1,2, Yujun Wang3, Xingzhi Xu4,5, Zhong-Wei Zhou *,1 and Zhao-Qi Wang *,1,6 1Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany 2Institute of Aging Research, School of Medicine, Hangzhou Normal University, Hangzhou, China 3Division of Biology, City of Hope National Medical Center/Beckman Research Institute, Duarte, CA, USA 4Beijing Key Laboratory of DNA Damage Response, College of Life Sciences, Capital Normal University, Beijing, China 5Guangdong Key Laboratory for Genome Stability & Disease Prevention, Shenzhen University School of Medicine, Shenzhen, Guangdong, China 6Faculty of Biology and Pharmacy, Friedrich-Schiller University of Jena, Jena, Germany *Corresponding author. Tel: +49 3641 656420; E-mail: [email protected] *Corresponding author. Tel: +49 3641 656415; Fax: +49 3641 656413; E-mail: [email protected] EMBO J (2017)36:3666-3681https://doi.org/10.15252/embj.201694443 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mutations of microcephalin (MCPH1) can cause the neurodevelopmental disorder primary microcephaly type 1. We previously showed that MCPH1 deletion in neural stem cells results in early mitotic entry that distracts cell division mode, leading to exhaustion of the progenitor pool. Here, we show that MCPH1 interacts with and promotes the E3 ligase βTrCP2 to degrade Cdc25A independent of DNA damage. Overexpression of βTrCP2 or the knockdown of Cdc25A remedies the high mitotic index and rescues the premature differentiation of Mcph1-deficient neuroprogenitors in vivo. MCPH1 itself is degraded by APC/CCdh1, but not APC/CCdc20, in late mitosis and G1 phase. Forced MCPH1 expression causes cell death, underlining the importance of MCPH1 turnover after mitosis. Ectopic expression of Cdh1 leads to premature differentiation of neuroprogenitors, mimicking differentiation defects of Mcph1-knockout neuroprogenitors. The homeostasis of MCPH1 in association with the ubiquitin-proteasome system ensures mitotic entry independent of cell cycle checkpoint. This study provides a mechanistic understanding of how MCPH1 controls neural stem cell fate and brain development. Synopsis Microcephalin (MCPH1) is mutated in the human neurodevelopmental disorder primary microcephaly type 1 (MCPH1), which is characterized by smaller-than-normal brain size. MCPH1 plays important roles in DNA damage response and cell cycle progression, which dictate proper neuroprogenitor fate. MCPH1 regulates βTrCP2-mediated Cdc25A degradation in mitotic entry. MCPH1 is degraded by APC/CCdh1 in late M and G1. The cell cycle-dependent homeostasis of MCPH1 ensures proper neurogenesis. Introduction Human primary microcephaly (MCPH, OMIM251200) is an autosomal recessive neurodevelopmental disorder, which is characterized by a marked reduction in brain size with a normal architecture and non-progressive mental retardation (Roberts et al, 2002). So far, 12 gene loci have been identified to be responsible for MCPH (reviewed by Kaindl, 2014), 10 of which encode proteins that are associated with centrosome or mitotic spindle poles, which function in controlling cell division (Hussain et al, 2013; Chavali et al, 2014; Mirzaa et al, 2014). Microcephalin (MCPH1), encoded by MCPH1, is a scaffold protein and is responsible for type I MCPH. MCPH1 contains three breast cancer carboxyl terminal (BRCT) domains, one in the N-terminus and the other two are tandem arranged in the C-terminus (Yu et al, 2003; Lin et al, 2010). The N-terminal BRCT domain of MCPH1 mediates its centrosome localization after DNA damage and is required for binding to the SWI/SNF complex to regulate DNA repair (Peng et al, 2009). The two C-terminal BRCT domains bind to γ-H2AX and are necessary for the recruitment of BRCA2/RAD51 to the damage sites for the execution of homologous recombination (HR) repair (Jeffers et al, 2008; Lin et al, 2010). Additionally, the C-terminal BRCTs interact with E2F1, which promotes the transcription of DNA repair genes, such as BRCA1 and CHK1 (Yang et al, 2008). MCPH1 also interacts, through its central region, with condensin II, which is thought to prevent premature chromosome condensation (PCC), a characteristic of MCPH1 cells (Trimborn et al, 2006). Human MCPH1 patient cells show a defective G2-M checkpoint, which is characterized by an impaired degradation of Cdc25A and hypo-phosphorylated Cdk1 due to a defective ATR-CHK1 activation (Xu et al, 2004; Lin et al, 2005; Alderton et al, 2006; Rai et al, 2006; Wood et al, 2008). We have generated a mouse model for MCPH1 with a null mutation of Mcph1 (Mcph1-del). These mutant mice exhibit microcephaly, which is characterized by a reduced thickness of the neocortex at birth, mimicking human MCPH1 patients (Gruber et al, 2011; Zhou et al, 2013). The genetic ablation of Mcph1 causes a premature differentiation of neuroprogenitors, thereby affecting spindle alignment and shifting the neuroprogenitor division mode from symmetric to asymmetric (Gruber et al, 2011). The hypo-phosphorylation of Cdk1 is found in Mcph1-del neuroprogenitors and correlates with a premature mitotic entry. The DNA damage response (DDR) function of MCPH1 cannot explain in full its physiological role in neurogenesis (Gruber et al, 2011; Zhou et al, 2013). Thus, the function of MCPH1 in regulating the G2-M checkpoint is believed to be responsible for the microcephaly phenotype. Two major ubiquitin E3 ligase complexes are implicated in cell cycle progression: the Skp1-Cul1-F-box (SCF) complex and the anaphase-promoting complex/cyclosome (APC/C) complex (Vodermaier, 2004). The β-transducin repeat-containing protein (βTrCP) is one of the F-box proteins that are responsible for the recruitment of specific substrates into the SCF complex (Suzuki et al, 1999). In mammals, there are two paralogs of βTrCP, namely βTrCP1 and βTrCP2 (also known as BTRC/FBXW1 and FBXW11, respectively; Suzuki et al, 1999). Both βTrCPs can degrade Cdc25A in response to DNA damage (Busino et al, 2003), which requires primed phosphorylation on Ser76 by CHK1 (Busino et al, 2003; Jin et al, 2003). APC/C is another ubiquitin E3 ligase complex, which has two activators, Cdc20 and Cdh1. While APC/CCdc20 plays a role in the early phase of mitosis and in metaphase/anaphase transition, APC/CCdh1 is activated in the mitotic exit and the early G1 phase to ensure G1 phase progression and to prevent S-phase entry (van Leuken et al, 2008; Meghini et al, 2016). It is also shown that a deletion of Cdh1 delays neurogenesis (van Leuken et al, 2008; Delgado-Esteban et al, 2013). In this report, we discover that MCPH1 directly regulates the Cdc25A stability through an interaction with and the promotion of the dimerization of βTrCP2 to ensure mitotic entry, but MCPH1 itself is degraded in late M phase and mainly in G1 phase in order to prevent cell death. The concerted action of Cdh1-MCPH1-βTrCP2-Cdc25A by the ubiquitin-proteasome system (UPS) decides neuroprogenitor fate and thus, provides a key mechanism for brain development. Results The identification of the MCPH1 interacting partners by a yeast two-hybrid (Y2H) screen Studies using MCPH1 mutant cellular and mouse models have shown an alteration of the G2-M transition and neuroprogenitor differentiation process after the MCPH1 inactivation (Xu et al, 2004; Lin et al, 2005; Alderton et al, 2006; Rai et al, 2006; Wood et al, 2008). Since MCPH1 is a scaffold protein, we hypothesized that its role in neuro-stem cell fate determination is thus likely through its effectors or partners. To search for these interaction partners, we carried out a yeast two-hybrid (Y2H) screen to identify novel MCPH1 partners by using a cDNA fragment encoding a polypeptide of the 96–612AA of human MCPH1 without all three BRCT domains as the bait. This would greatly reduce the number of MCPH1 interactors that are functionally associated with DDR. A total of 99 LacZ-positive colonies were obtained from three independent screens. Seventy-seven coding sequences from 66 genes with known functions were confirmed by sequencing (Table EV1). Among these coding genes, we focused on FBXW11 that encodes βTrCP2, which is a WD40 domain-containing F-box protein responsible for substrate binding in the SCF ubiquitin E3 ligase complex. MCPH1 interacts with βTrCP2 in vivo To verify the interaction between MCPH1 and βTrCP2, HA-tagged MCPH1 (HA-MCPH1) and FLAG-tagged βTrCP2 (FLAG-βTrCP2) or βTrCP1 (FLAG-βTrCP1) were co-transfected into 293T cells. Co-immunoprecipitation (Co-IP) assays revealed that HA-MCPH1 interacted strongly and specifically with FLAG-βTrCP2, but negligibly with FLAG-βTrCP1 (Fig 1A). This interaction was further confirmed by Co-IP showing that ectopic expressed FLAG-βTrCP2 (Fig 1B) or endogenous βTrCP2 (Fig 1C) was present in the endogenous MCPH1 immunocomplex (Fig 1B and C). To determine whether MCPH1 also binds to other WD40 domain-containing F-box proteins or to other components of the SCF complex, we co-transfected HA-MCPH1 with FLAG-tagged FBW7, Skp2, or Skp1 into 293T cells and found that MCPH1 bound mainly to βTrCP2 (Fig 1D). Because MCPH1 is involved in DDR (Xu et al, 2004; Lin et al, 2010), we next asked whether the interaction between MCPH1 and βTrCP2 is DDR-dependent. Co-IP assays were carried out at the different time point after treating cells with a low does (2 Gy) and a high does (10 Gy) of ionizing radiation (IR). A low dose (2 Gy) of IR did not change the MCPH1 level till 24 h after irradiation (Fig EV1A). However, 10 Gy IR reduced the MCPH1 protein level, which correlates well with DDR, judged by the super-shifted Chk2, which is an indication of phosph-Chk2 (Fig 1E). However, neither dose of IR disrupts the interaction between MCPH1 and βTrCP2 in the Co-IP (Figs 1E and EV1A). These data suggest that MCPH1 interacts with βTrCP2 in vivo under unperturbed conditions and also upon DNA damage. Figure 1. Interaction of MCPH1 with βTrCP2 HA-MCPH1 interacted with FLAG-βTrCP2. HA-MCPH1 was co-transfected with FLAG-βTrCP1 or FLAG-βTrCP2 into 293T cells. Immunoprecipitation (IP) was performed using anti-FLAG antibody, and immunoblotting (IB) was performed using anti-FLAG or anti-HA antibody. The experiment was repeated twice. Co-IP assay of endogenous MCPH1 was performed using an anti-MCPH1 antibody in 293T cells after transfection with FLAG-βTrCP1 or FLAG-βTrCP2. The experiment was repeated twice. Endogenous MCPH1 interacts with βTrCP2 in Neuro2A cells. IP was performed using an anti-MCPH1 antibody, and IB was performed using anti-MCPH1 or anti-βTrCP2 antibody. HA-MCPH1 was co-transfected with FLAG-tagged indicated F-box protein constructs. IP and IB were performed using anti-HA or FLAG antibody as indicated. Neuro2A cells were treated with 10 Gy ionizing radiation (IR) with or without the proteasome inhibitor MG132 and harvested at the indicated time after IR. Endogenous Co-IP was performed using an anti-MCPH1 antibody, and IB was performed using an anti-MCPH1 or anti-βTrCP2 antibody. The experiment was repeated twice. Left panel: Schematic diagram of full-length and deletion mutants of MCPH1. Red boxes represent the BRCT domain. FL: 1–835aa, ∆BR1: 94–835aa, ∆BR2: ∆671–730aa, ∆BR3: 1–730aa, ∆BR2-3: 1–670aa. Right panel: HA-tagged full-length and deletion mutants of MCPH1 were co-transfected with FLAG-βTrCP2. IP and IB were performed using the anti-FLAG or anti-HA antibody. The experiment was repeated twice. Left panel: Schematic diagram of βTrCP2 full-length and a series of deletion mutants. The yellow box represents the D domain, the gray box represents the F-box domain, and purple box represents the WD domain. FL: 1–529aa, ∆N: 121–529aa, ∆F: ∆129–167aa, ∆C: 1–237aa. Right panel: HA-MCPH1 was co-transfected with indicated βTrCP2 deletion mutants. IP and IB were performed using anti-FLAG or anti-HA antibody. Input in each panel is 10% of total cell lysates. The FLAG-EV or HA-EV blots are not shown because their size is too small to be included. The number under each sample is a ratio to the FL sample after normalization to Input of the displayed blots. Asterisk marks the IgG band. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. MCPH1 interacts with βTrCP2 Neuro2A cells were treated with low dose (2 Gy) IR and harvested at the indicated time after IR. An endogenous Co-IP was performed using an anti-MCPH1 antibody and IB was performed using an anti-MCPH1 or anti-βTrCP2 antibody. HA-MCPH1 was co-transfected with FLAG-EV or FLAG-βTrCP2 into 293T cells. The MCPH1 protein level of was examined by using anti-HA antibody. β-actin was used to control loading. The level of MCPH1 (IB:HA) after normalization to β-actin is presented as a ratio to HA-MCPH1/FLAG-EV of the displayed blots. The knockdown efficiency by the two vectors against shβTrCP1 or shβTrCP2 was examined by using an anti-FLAG antibody in 293T cells which were transfected with FLAG-βTrCP1 or FLAG-βTrCP2. GAPDH was used to control the loading. The level of FLAG-βTrCP1 or FLAG-βTrCP2 after the normalization to GAPDH is presented as a ratio to shLuc of the displayed below the blots. shRNA against MCPH1 or βTrCP2 was transfected into HeLa cells. The protein level of endogenous MCPH1 and βTrCP2 was analyzed by IB using an anti-MCPH1 or anti-βTrCP2 antibody, respectively. β-actin is used as a loading control. The level is quantified as a ratio to shLuc after the normalization of β-actin of the blots on display. The experiment was repeated twice. HA-MCPH1 was co-transfected with FLAG-EV or FLAG-βTrCP2 into 293T cells. The cells were treated with 10 Gy IR and recovered for the indicated time, then treated with or without with MG132 for 3 h. The MCPH1 level was analyzed by using an anti-HA antibody, and the βTrCP2 level was monitored by an anti-FLAG antibody. β-actin was used to control the loading. Co-transfection of HA-Cdc25B with FLAG-EV or FLAG-βTrCP2 into 293T cells transfected with shLuc or shMCPH1-1. The Cdc25B level was examined by an anti-HA antibody, and the βTrCP2 level is monitored by an anti-FLAG antibody. GAPDH was used to control loading. Western blot analysis of shLuc- or shMCPH1-1-transfected HeLa cells, which were subsequently knocked down for Cdc25A or Cdc25B (positive control), or overexpressed FLAG-βTrCP2. β-actin was used to control the loading. The levels of Cdc25A and Cdc25B are quantified as a ratio to shLuc after the normalization of β-actin of the blots on display. The experiment was repeated twice. The FLAG-EV or HA-EV blots are not shown because their size is too small to be included. IB analysis of the knockdown efficiency of two shCdc25A vectors or siCdc25A using an anti-HA antibody after co-transfection with HA-Cdc25A. β-actin was used to control the loading. IB analysis of knockdown efficiency of two shCdc25B vectors or siCdc25B using an anti-HA antibody after co-transfection with HA-Cdc25B. β-actin was used to control the loading. Download figure Download PowerPoint We next mapped the interacting regions among MCPH1 and βTrCP2. To this end, different truncations of MCPH1 and βTrCP2 were prepared (Fig 1F and G). A Co-IP analysis showed that βTrCP2 bound to wild-type MCPH1 as well as mutant MCPH1 with a deletion of individual or all BRCT domains (Fig 1F). Thus, BRCT domains are not necessary for the interaction, which is consistent with the original Y2H data. The interaction most likely takes place in the middle part of MCPH1. We then investigated which domain of βTrCP2 is responsible for the interaction with MCPH1. An IP assay revealed that the interaction between MCPH1 and βTrCP2 was not affected by truncating βTrCP2 neither in the N-terminus (ΔN), which includes the D domain that is required for dimer formation, nor in the F-box domain (ΔF) that binds to Skp1, although these truncations showed less affinity to MCPH1 compared to full-length βTrCP2 (Fig 1G). Of note, the βTrCP2 truncation without a C-terminal WD40-containing domain, which is required for substrate binding, completely lost the ability to pull down MCPH1 (Fig 1G), suggesting that MCPH1 may be a substrate of βTrCP2 or may antagonize other substrates of βTrCP2. MCPH1 is not a substrate for βTrCP2, but stimulates its activity to degrade Cdc25A Next, we examined whether SCFβTrCP2 degrades MCPH1. The MCPH1 level was not affected when it was co-expressed with βTrCP2 in 293T cells (Figs 1A, B, D and F, and EV1B). Moreover, knockdown of βTrCP2 (Fig EV1C) did not increase the endogenous MCPH1 level (Fig EV1D). Since 10 Gy IR reduced endogenous MCPH1 as well as βTrCP2 (see Fig 1E), we wondered whether MCPH1 degradation was mediated by βTrCP2 in DDR. To test this, cells ectopically expressing MCPH1 and βTrCP2 were exposed to 10 Gy of IR. No obvious reduction in the level of MCPH1 found in βTrCP2 expressing cells, compared to EV-transfected samples after 10 Gy IR treatments (Fig EV1E). Thus, MCPH1 is most likely not a substrate of βTrCP2 under either unperturbed or DNA damage conditions. To further address the meaning of the interaction between MCPH1 and βTrCP2, we tested whether this interaction modulates the βTrCP2 activity by examining the status of SCFβTrCP2 substrates Cdc25B and Cdc25A (Busino et al, 2003; Thomas et al, 2010; Young & Pagano, 2010). While we did not observe an obvious reduction of Cdc25B when co-transfected with βTrCP2 (Fig EV1F and G, lanes 3 and 8), an overexpression of βTrCP2 reduced the Cdc25A level to a level similar to shCdc25A (Fig EV1G, lanes 3 and 4). Interestingly, when we knocked down MCPH1 by shRNA, we detected a higher level of endogenous as well as of ectopically expressed Cdc25A compared to shLuc controls (Figs 2A and B, and EV1G), suggesting that Cdc25A is a target of MCPH1-βTrCP2 interaction. Figure 2. MCPH1 regulates βTrCP2 to degrade Cdc25A Two shRNAs against MCPH1 (shMCPH1-1 and shMCPH1-3) were transfected into 293T cells. The protein level of endogenous MCPH1 and Cdc25A was analyzed by IB using indicated antibodies, respectively. β-actin is used as a loading control. The level of MCPH1 and Cdc25A is a ratio to shLuc after normalization of β-actin of the blots on display. The experiment was repeated three times. HA-Cdc25A and FLAG-βTrCP2 were co-transfected into shLuc- or shMCPH1-1-transfected 293T cells. The degradation of Cdc25A (HA) and interaction with βTrCP2 (FLAG) were investigated using the anti-HA antibody. β-actin is used as a loading control. The level of Cdc25A (IB:HA) in Input is normalized to β-actin and shLuc/HA-Cdc25A is set as 1.0. The number under each sample in the immunocomplex (IP:FLAG) is a ratio to shLuc/βTrCP2/Cdc25A (IB:HA) after normalization to Input (IB:HA) of the blots on display. The experiment was repeated twice. GFP-MCPH1, FLAG-βTrCP2, and HA-Cdc25A were co-transfected into 293T cells. IP was performed using anti-FLAG antibody. IB was performed using anti-FLAG, anti-HA, or anti-GFP antibody. β-actin is used as a loading control. Input in each panel is 10% of total cell lysates. The level of Cdc25A (in Input IB:HA) is a ratio to HA-Cdc25A (in GFP-EV/FLAG-EV transfected, lane 2) after normalization to β-actin of the displayed blots. The stability of HA-Cdc25A was examined after cycloheximide (CHX) treatment for the indicated time in shLuc- and shMCPH1-1-transfected 293T cells and visualized by an anti-HA antibody. β-actin was used to control the loading. The level of HA-Cdc25A after normalization to β-actin is presented as a ratio to untreated shLuc/HA-Cdc25A or shMCPH1/HA-Cdc25A of the blots on display. The experiment was repeated three times. Dimer formation of βTrCP2 was examined by co-transfection of HA-βTrCP2 with FLAG-βTrCP1 or FLAG-βTrCP2 in shLuc- or shMCPH1-1-transfected cells. IP was performed using anti-FLAG antibody, and IB was performed using anti-FLAG or anti-HA antibody. The number under each sample in the immunocomplex (IP:FLAG) is a ratio to βTrCP2:βTrCP1 heterodimer (IB:HA) after normalization to Input (IB:HA) of displayed blots. HA-Cdc25A and FLAG-βTrCP2 were co-transfected into shLuc- or shMCPH1-1-transfected 293T cells. The degradation of Cdc25A was investigated using the anti-HA antibody. The cells were treated with CHK1 inhibitor (UCN-01) before harvest. β-actin is used as a loading control. The level of Cdc25A (IB:HA) is a ratio to shLuc/HA-Cdc25A after normalization to β-actin. The experiment was repeated three times. Asterisk marks a non-specific band recognized by the anti-MCPH1 antibody. HA-Cdc25A-S76D and FLAG-βTrCP2 were co-transfected into shLuc- or shMCPH1-1-transfected 293T cells. The degradation of Cdc25A-S76D was investigated by using anti-HA antibody. β-actin is used as a loading control. The level of Cdc25A is a ratio to shLuc/Cdc25A-S76D (IB:HA) after normalization to β-actin of the blots on display. The FLAG-EV or HA-EV blots are not shown because their size is too small to be included. The experiment was repeated twice. Download figure Download PowerPoint To further confirm these findings, HA-Cdc25A and FLAG-βTrCP2 were co-transfected with or without the shMCPH1 expression vectors into 293T cells. The HA-Cdc25A levels were greatly reduced in the cells co-expressing FLAG-βTrCP2, as compared to the FLAG-EV (empty vector) control (Fig 2B, Input, lanes 1 and 2). The knockdown of MCPH1 by shRNA led to a high level of Cdc25A (Fig 2B, Input, lanes 1 and 4) and strongly suppressed βTrCP2-mediated Cdc25A degradation (Fig 2B, Input, lanes 2 and 5). In both cases, Cdc25A was further stabilized by a treatment with a proteasome inhibitor MG132 (Fig 2B, Input, lanes 2 and 3, lanes 5 and 6). Reversely, an overexpression of MCPH1 decreased the Cdc25A level (Fig 2C, lane 4), which was further enhanced by an ectopic expression of βTrCP2 (Fig 2C, lane 5). Next, we measured the stability and kinetics of Cdc25A in shMCPH1-transfected cells using cycloheximide (CHX), a protein synthesis inhibitor. While Cdc25A was gradually degraded during the time course of the CHX treatment in control shLuc-transfected 293T cells, it stayed at a high level in shMCPH1 cells (Figs 2D, and EV2A and B), suggesting that the Cdc25A degradation is dependent on MCPH1. To further identify during which cell cycle phase MCPH1-mediated degradation of Cdc25A occurs, we measured the stability and kinetics of endogenous Cdc25A in S (1 and 4 h) and G2 (6 h) phases after releasing from double-thymidine (T-T) block which synchronized cells in the early S-phase, together with the CHX treatment (Fig EV2C and D). The increased stability of Cdc25A after MCPH1 deletion was mainly found in G2 phase (Fig EV2C). At each time point, we also analyzed Cdc25A mRNA and found that Cdc25 mRNA was relatively lower compared to controls (Fig EV2E), ruling out that the increased Cdc25A protein in shMCPH1 cells is due to the Cdc25A transcription. In addition, the increased stability of Cdc25A in G2 phase was also observed after βTrCP2 knockdown (Fig EV2F and G), which is similar to that after MCPH1 deletion. Taken together, these data suggest that MCPH1 regulates mitotic entry in G2 phase via a βTrCP2-mediated degradation of Cdc25A. Click here to expand this figure. Figure EV2. MCPH1 regulates degradation of Cdc25A in G2 phase The stability of endogenous Cdc25A was examined after cycloheximide (CHX) treatment for the indicated time in shLuc- or shMCPH1-1-transfected 293T cells. β-actin is used as a loading control. The diagrams of Cdc25A stability was shown in the right panel after normalization to β-actin as a ratio to untreated shLuc or shMCPH1-1 of the blots on display. The experiment was repeated twice. The stability of FLAG-Cdc25A was examined after cycloheximide (CHX) treatment for the indicated time in shLuc- or shMCPH1-3-transfected 293T cells. β-actin is used as a loading control. The experiment was repeated twice. Asterisk marks a non-specific band recognized by the anti-MCPH1 antibody. Western blot analysis of the stability of endogenous Cdc25A after cycloheximide (CHX) treatment for the indicated time in shLuc- and shMCPH1-1-transfected 293T cells at different time points after double-thymidine (T-T) block. Lamin B1 was used to control the loading. The level of Cdc25A after normalization to lamin B1 is presented as a ratio to untreated shLuc of the blots on display. The experiment was repeated three times. Asterisk marks a non-specific band recognized by the anti-MCPH1 antibody. Cell cycle profile, analyzed by flow cytometry, of cells used for panel (C) at different time points after the T-T block release. mRNA level of Cdc25A and MCPH1 was analyzed by qRT–PCR at indicated time points after release from the T-T block. The experiment was repeated twice. Western blot analysis of the stability of endogenous Cdc25A after cycloheximide (CHX) treatment for the indicated time in shLuc-, shβTrCP2-, or shMCPH1-transfected 293T cells at 6 h after double-thymidine (T-T) block. The upper β-actin blot controls the loading of Cdc25A and the lower β-actin blot controls MCPH1 and βTrCP2 loading. The quantification of Cdc25A, MCPH1, and βTrCP2 is performed by normalization to the corresponding β-actin blot and presented as a ratio to CHX untreated shLuc under each lane. The experiment was repeated twice and the quantification is displayed below the blots. Asterisk marks a non-specific band recognized by the anti-MCPH1 antibody. Cell cycle profile, analyzed by flow cytometry, of cells used for panel (F) at 6 h after the T-T block release. HA-βTrCP2 was co-transfected with FLAG-Skp1 into shLuc- or shMCPH1-1-transfected cells. IP and IB were performed by using the anti-HA or anti-FLAG antibody, respectively. β-actin is used as a loading control. Download figure Download PowerPoint To address how MCPH1 promotes Cdc25A degradation, we tested whether MCPH1 deletion would affect the binding between Cdc25A and βTrCP2. Since MCPH1 binds to the substrate interacting domain WD40 of βTrCP2, this may compromise the recruitment of Cdc25A to βTrCP2 for degradation. However, neither knockdown (Fig 2B, IP:FLAG, lanes 2 and 5) nor overexpression (Fig 2C, IP:HA, lanes 8 and 10) of MCPH1 obviously impaired the interaction between βTrCP2 and Cdc25A, indicating that MCPH1 does not mediate the interaction between Cdc25A and βTrCP2. This was further supported by the lack of direct interaction between MCPH1 and Cdc25A (Fig 2C, lanes 9 and 10). Moreover, the knockdown of MCPH1 did not affect the interaction between βTrCP2 and Skp1, the linker of βTrCP2 to the SCF complex (Fig EV2H). Like other WD domain-containing F-box proteins, while the monomer form harbors the ubiquitination activit
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