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SPOP-mediated poly-ubiquitination of Geminin inhibits MCM proteins binding to Cdt1 a PC-3 cells were infected with lentivirus expressing indicated shRNAs or WT or mutated Geminin and harvested for co-IP and WB analysis. b PC-3 cells were infected with lentivirus expressing empty vector or different SPOP mutants and harvested for co-IP and WB. c The structure of human Cdt1-Geminin is overlaid to that of yeast Cdt1-MCM after conformational optimization. A modeled K27-linked poly-ubiquitin chain attached to Geminin Lys127 (light blue) is incompatible with Cdt1-Geminin binding to the MCM complex. d–f PC-3 cells were infected with lentivirus expressing control shRNA or Geminin-specific shRNA in combination with empty vector (EV), Geminin WT, or K100/127R mutant. Cells were pulse-labeled with 30 μM BrdU prior to WB analysis (d), FACS analysis (e), or quantification (f). Data are presented as the mean ± SD of three independent experiments. Two-tailed unpaired Student’s t-test; ***P < 0.001. Source data are provided in this paper or Mendeley database (10.17632/8n7xt5rkhc.1). Similar results for (a), (b), and (d) panels were obtained in three independent experiments.
Source publication
Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitina...
Contexts in source publication
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... DNA replication firing 20,21 , we further investigated whether SPOP-mediated Geminin ubiquitination impacts MCM complex access to Cdt1. As expected, Geminin knockdown increased Cdt1 binding with pre-RC proteins such as MCM2, CDC6, and ORC2, and this process was reversed by expression of shRNA-resistant WT Geminin but not the K100R/K127R mutant ( Fig. 4a and Supplementary Fig. 3d). Similar to the effect of the ubiquitination-resistant mutant of Geminin, expression of cancer-derived SPOP mutants also increased Cdt1 binding to MCM2, CDC6, and ORC2 but had no influence on Geminin binding to Cdt1 ( Fig. 4b and Supplementary Fig. 3e). Current structural knowledge of the replication origin ...Context 2
... was reversed by expression of shRNA-resistant WT Geminin but not the K100R/K127R mutant ( Fig. 4a and Supplementary Fig. 3d). Similar to the effect of the ubiquitination-resistant mutant of Geminin, expression of cancer-derived SPOP mutants also increased Cdt1 binding to MCM2, CDC6, and ORC2 but had no influence on Geminin binding to Cdt1 ( Fig. 4b and Supplementary Fig. 3e). Current structural knowledge of the replication origin and origin recognition complex formation is mostly derived from studies in budding yeast 46,47 . A single-particle cryo-EM structure of a pre-insertion loading intermediate of ORC-Cdc6-Cdt1-MCM (OCCM) complex from yeast was recently obtained by ...Context 3
... structure revealed large amplitude motions of the C-terminal helical regions of the Geminin homodimer construct used for crystallography (Supplementary Fig. 3h). Taking this flexibility into account, we adjusted and optimized the conformation of Geminin C-terminal regions to eliminate any steric clash. Our structural model for Gemininbound OCCM (Fig. 4c) predicts that K27-linked poly-ubiquitination at Geminin Lys127 would cause a steric clash with the MCM complex that would displace Cdt1 from MCM. This analysis provides a plausible explanation as to how SPOP-mediated ubiquitination of Geminin affects the Cdt1-MCM interaction, while not directly affecting the Geminin-Cdt1 interaction. ...Context 4
... poly-ubiquitination at Geminin Lys127 would cause a steric clash with the MCM complex that would displace Cdt1 from MCM. This analysis provides a plausible explanation as to how SPOP-mediated ubiquitination of Geminin affects the Cdt1-MCM interaction, while not directly affecting the Geminin-Cdt1 interaction. In agreement with our model in Fig. 4c, co-IP and mass spectrometry assays showed that Geminin could pull down MCM proteins and that SPOP-mediated poly-ubiquitination of Geminin reduced Geminin-MCM complex formation ( Supplementary Fig. 3i-k). In addition, in vitro translated MCM2 protein showed no direct binding to GST-Geminin purified from bacteria ( Supplementary Fig. 3l ...Context 5
... in PC-3 cells and the increase was reversed by restored expression of shRNA-resistant WT Geminin, but not by the K100R/K127R double mutant ( Fig. 4d-f). Together, these data suggest that SPOP-mediated Geminin poly-ubiquitination is important for Geminin inhibition of the replication function of ...Context 6
... determine the impact of cell cycle-dependent oscillation of SPOP protein on Geminin poly-ubiquitination, we synchronized PC-3 cells expressing HA-tagged K27-only ubiquitin (Ub) (HAUb-K27) by nocodazole ( Supplementary Fig. 4a). A dramatic increase in Geminin K27-linked poly-ubiquitination was observed in control cells at S and early G2 phases compared to cells at other phases (Fig. 5d). ...Context 7
... the cell-cycledependent K27-linkage poly-ubiquitination of Geminin was completely abolished in SPOP-F133V mutant-expressing PC-3 cells even though Geminin protein level retained a similar oscillation pattern as in control cells (Fig. 5d). Notably, the total poly-ubiquitination of Geminin was also decreased at S and early G2 phases in F133V mutant cells transfected with WT ubiquitin (Supplementary Fig. 4b). Furthermore, FACS analysis with BrdU- labeled cells showed that SPOP-F133V expression resulted in an increase of re-replicated cells (>4N) in PC-3 cells (Fig. 5e, f and Supplementary Fig. 4c). ...Context 8
... the total poly-ubiquitination of Geminin was also decreased at S and early G2 phases in F133V mutant cells transfected with WT ubiquitin (Supplementary Fig. 4b). Furthermore, FACS analysis with BrdU- labeled cells showed that SPOP-F133V expression resulted in an increase of re-replicated cells (>4N) in PC-3 cells (Fig. 5e, f and Supplementary Fig. 4c). A similar result was obtained in Geminin knockdown cells regardless of SPOP-F133V expression (Fig. 5e, f). ...Context 9
... a positive control, we found that the distance between two adjacent origins (inter-origin distance) was decreased in cells treated with VE-822 ( Fig. 5g and Supplementary Fig. 4d). However, this phenomenon was also observed in Geminin knockdown or SPOP-F133V mutant-expressing cells without VE-822 treatment (Fig. 5g). Importantly, the inter-origin distance in F133V and/or Geminin knockdown cells was lower than that in control cells and was further reduced after co-treatment of VE-822 (Fig. 5g). The decreased ...Context 10
... decreased inter-origin distance indicates that there is increased origin firing in SPOP-mutated cells, providing a plausible explanation as to why there is increased binding of pre-RC proteins to chromatin (Fig. 5b). DNA re-replication also increased by several folds in PC-3 cells expressing SPOP mutant or Geminin shRNA compared to control cells ( Fig. 5h and Supplementary Fig. 4e), and their effects were further enhanced by ATR inhibitor treatment (Fig. 5h). Both increased replication stress and re-replication burden sensitize ATR inhibition-caused replication catastrophe and double-strand breaks and ultimately lead to cell death 54 . ...Context 11
... data suggest that SPOP and Geminin work in the same pathway in prohibition of ATR inhibition-caused replication catastrophe and double-strand breaks. We also checked the numbers of intrachromosomal breaks per cell after these groups of cells were treated or not with ATR inhibitor ( Fig. 5j and Supplementary Fig. 4e, f). The results were consistent with changes in γ-H2AX level (Fig. 5i). ...Citations
... Liquid chromatography-mass spectrometry (LC-MS) analysis was performed as described previously 75 Benjamini and Hochberg's approach for controlling the false discovery rate, the genes meeting the criteria of a P value < 0.05, an adjusted P value < 0.05 and a fold change >=1.5 were considered to be differentially expressed genes. ...
CDK4/6 inhibitors (CDK4/6i) show anticancer activity in certain human malignancies, such as breast cancer. However, their application to other tumor types and intrinsic resistance mechanisms are still unclear. Here, we demonstrate that MYC amplification confers resistance to CDK4/6i in bladder, prostate and breast cancer cells. Mechanistically, MYC binds to the promoter of the E3 ubiquitin ligase KLHL42 and enhances its transcription, leading to RB1 deficiency by inducing both phosphorylated and total pRB1 ubiquitination and degradation. We identify a compound that degrades MYC, A80.2HCl, which induces MYC degradation at nanomolar concentrations, restores pRB1 protein levels and re-establish sensitivity of MYC high-expressing cancer cells to CDK4/6i. The combination of CDK4/6i and A80.2HCl result in marked regression in tumor growth in vivo. Altogether, these results reveal the molecular mechanisms underlying MYC-induced resistance to CDK4/6i and suggest the utilization of the MYC degrading molecule A80.2HCl to potentiate the therapeutic efficacy of CDK4/6i.
... The MATH domain of SPOP selectively recognized and recruited substrates, such as c-Myc,15 Daxx,16 BRD4,17 and PD-L1,21 which is identical to our results. In addition, most SPOP mutations are also detected in the domain.17,19,44 Our results demonstrated the distinct effect of MATH domain on SPOPmediated anti-HBV function. ...
Hepatitis B virus (HBV) infection remains a significant public health burden worldwide. The persistence of covalently closed circular DNA (cccDNA) within the nucleus of infected hepatocytes is responsible for the failure of antiviral treatments. The ubiquitin proteasome system (UPS) has emerged as a promising antiviral target, as it can regulate HBV replication by promoting critical protein degradation in steps of viral life cycle. Speckle-type POZ protein (SPOP) is a critical adaptor for Cul3-RBX1 E3 ubiquitin ligase complex, but the effect of SPOP on HBV replication is less known. Here, we identified SPOP as a novel host antiviral factor against HBV infection. SPOP overexpression significantly inhibited the transcriptional activity of HBV cccDNA without affecting cccDNA level in HBV-infected HepG2-NTCP and primary human hepatocyte cells. Mechanism studies showed that SPOP interacted with hepatocyte nuclear factor 1α (HNF1α), and induced HNF1α degradation through host UPS pathway. Moreover, the antiviral role of SPOP was also confirmed in vivo. Together, our findings reveal that SPOP is a novel host factor which inhibits HBV transcription and replication by ubiquitination and degradation of HNF1α, providing a potential therapeutic strategy for the treatment of HBV infection. K E Y W O R D S hepatitis B virus, HNF1α, host factor, SPOP, ubiquitination J Med Virol. 2023;95:e29254. wileyonlinelibrary.com/journal/jmv
... The CRL3-SPOP complex catalyzes both degradative and non-degradative ubiquitination depending on the substrate. For example, CRL3-SPOP catalyzes K27-linked non-degradative ubiquitination of Geminin, which prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex The p-values were calculated using the One-way ANOVA test in a, c, d, f and the Two-way ANOVA test in b. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 [31]. CRL3-SPOP catalyzed mixed K6-, K27-, and K29linked non-degradative ubiquitination of p62, which suppresses p62-dependent autophagy [32]. ...
Background
The gene encoding the E3 ubiquitin ligase substrate-binding adapter Speckle-type BTB/POZ protein ( SPOP ) is frequently mutated in prostate cancer (PCa) and endometrial cancer (EC); however, the molecular mechanisms underlying the contribution of SPOP mutations to tumorigenesis remain poorly understood.
Methods
BRAF harbors a potential SPOP-binding consensus motif (SBC) motif. Co-immunoprecipitation assays demonstrated that BRAF interacts with SPOP. A series of functional analyses in cell lines were performed to investigate the biological significance of MAPK/ERK activation caused by SPOP mutations.
Results
Cytoplasmic SPOP binds to and induces non-degradative ubiquitination of BRAF, thereby reducing the interaction between BRAF and other core components of the MAPK/ERK pathway. SPOP ablation increased MAPK/ERK activation. EC- or PCa-associated SPOP mutants showed a reduced capacity to bind and ubiquitinate BRAF. Moreover, cancer-associated BRAF mutations disrupted the BRAF-SPOP interaction and allowed BRAF to evade SPOP-mediated ubiquitination, thereby upregulating MAPK/ERK signaling and enhancing the neoplastic phenotypes of cancer cells.
Conclusions
Our findings provide new insights into the molecular link between SPOP mutation-driven tumorigenesis and aberrant BRAF-dependent activation of the MAPK/ERK pathway.
... As both a negative and positive regulator of the pre-replication complex (pre-RC), geminin directly binds to chromatin licensing and DNA replication factor 1 (Cdt1) to regulate nuclear DNA initiation (Wohlschlegel James et al., 2000;Ballabeni et al., 2004;Lee et al., 2004). Aberrant expression or degradation of geminin ultimately triggers genomic instability and oncogenic transformation (Zhu & Depamphilis, 2009;Champeris Tsaniras et al., 2018;Ma et al., 2021). Oncogenic transformation contributes to reactive oxygen species (ROS) production (Park et al., 2014;Maya-Mendoza et al., 2015), which in turn influences the occurrence of replication stress through polymerase activity or the physical obstacle of oxidizing dNTPs and dissociation of peroxiredoxin2 oligomers (PRDX2) (Sedletska et al., 2013;Park et al., 2014;Maya-Mendoza et al., 2015;Somyajit et al., 2017;Meng et al., 2018). ...
Lysophosphatidic acid (LPA) and geminin are overexpressed in ovarian cancer, and increasing evidence supports their contribution to ovarian tumor development. Here, we reveal that geminin depletion induces autophagy suppression and enhances reactive oxygen species (ROS) production and apoptosis of high-grade serous ovarian cancer (HGSOC) cells. Bioinformatics analysis and pharmacological inhibition studies confirm that LPA activates geminin expression in the early S phase in HGSOC cells via the LPAR1/3/MMPs/EGFR/PI3K/mTOR pathway. Furthermore, LPA phosphorylates Aurora-A kinase on Thr288 through EGFR transactivation, and this event potentiates additional geminin stabilization. In turn, overexpressed and stabilized geminin regulates DNA replication, cell-cycle progression, and cell proliferation of HGSOC cells. Our data provide potential targets for enhancing the clinical benefit of HGSOC precision medicine.
... Geminin, a DNA replication inhibitor, is activated during cell cycle-progression by the anaphase-promotion complex (APC) leading to initiation of sister chromatid separation [77]. The specific role of geminin is to limit DNA replication and trigger sister chromatid separation and, therefore, it is considered to qualify exceptionally well as a cell cyclespecific proliferation marker [78][79][80]. ...
Purpose
Triple-negative breast cancer (TNBC) is an aggressive disease lacking specific biomarkers to guide treatment decisions. We evaluated the combined prognostic impact of clinical features and novel biomarkers of cell cycle-progression in age-dependent subgroups of TNBC patients.
Methods
One hundred forty seven TNBC patients with complete clinical data and up to 18 year follow-up were collected from Turku University Hospital, Finland. Eight biomarkers for cell division were immunohistochemically detected to evaluate their clinical applicability in relation to patient and tumor characteristics.
Results
Age at diagnosis was the decisive factor predicting disease-specific mortality in TNBC (p = 0.002). The established prognostic features, nodal status and Ki-67, predicted survival only when combined with age. The outcome and prognostic features differed significantly between age groups, middle-aged patients showing the most favorable outcome. Among young patients, only lack of basal differentiation predicted disease outcome, indicating 4.5-fold mortality risk (p = 0.03). Among patients aged > 57, the established prognostic features predicted disease outcome with up to 3.0-fold mortality risk for tumor size ≥ 2 cm (p = 0.001). Concerning cell proliferation, Ki-67 alone was a significant prognosticator among patients aged > 57 years (p = 0.009). Among the studied cell cycle-specific biomarkers, only geminin predicted disease outcome, indicating up to 6.2-fold increased risk of mortality for tumor size < 2 cm (p = 0.03).
Conclusion
Traditional clinical features do not provide optimal prognostic characterization for all TNBC patients. Young age should be considered as an additional adverse prognostic feature in therapeutic considerations. Increased proliferation, as evaluated using Ki-67 or geminin immunohistochemistry, showed potential in detecting survival differences in subgroups of TNBC.
... Speckle-type BTB/POZ protein (SPOP) is a substrate recognition receptor of the cullin-3 (CUL3)/RING-type ubiquitin E3 complex that has emerged as a gatekeeper of genome stability given its critical roles in DDR and DNA replication 59 . Recently, two studies further extended the role of SPOP in maintaining DNA integrity through non-proteolytic ubiquitylation and identified 53BP1 and Geminin as substrates of the CUL3/SPOP complex 60,61 (Fig. 2a). SPOP promotes the K27-linked nondegradative poly-ubiquitylation of the essential DNA replication protein Geminin during S phase and prevents DNA replication over-firing by inhibiting the interaction of the Geminin binding partner Cdt1 with the MCM (mini-chromosome maintenance) complex 60 . ...
... Recently, two studies further extended the role of SPOP in maintaining DNA integrity through non-proteolytic ubiquitylation and identified 53BP1 and Geminin as substrates of the CUL3/SPOP complex 60,61 (Fig. 2a). SPOP promotes the K27-linked nondegradative poly-ubiquitylation of the essential DNA replication protein Geminin during S phase and prevents DNA replication over-firing by inhibiting the interaction of the Geminin binding partner Cdt1 with the MCM (mini-chromosome maintenance) complex 60 . Cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitylation and lead to replication stress and cell death upon ATR kinase inhibition, implying that cancers with defective SPOP-Geminin signaling might be responsive to ATR inhibitors treatment. ...
Ubiquitylation is one of the most common post-translational modifications (PTMs) of proteins that frequently targets substrates for proteasomal degradation. However it can also result in non-proteolytic events which play important functions in cellular processes such as intracellular signaling, membrane trafficking, DNA repair and cell cycle. Emerging evidence demonstrates that dysfunction of non-proteolytic ubiquitylation is associated with the development of multiple human diseases. In this review, we summarize the current knowledge and the latest concepts on how non-proteolytic ubiquitylation pathways are involved in cellular signaling and in disease-mediating processes. Our review, may advance our understanding of the non-degradative ubiquitylation process.
DNA replication is a vulnerable cellular process, and its deregulation leads to genomic instability. Here, we demonstrate that chromobox protein homolog 3 (CBX3) binds replication protein A 32-kDa subunit (RPA2) and regulates RPA2 retention at stalled replication forks. CBX3 is recruited to stalled replication forks by RPA2 and inhibits ring finger and WD repeat domain 3 (RFWD3)–facilitated replication restart. Phosphorylation of CBX3 at serine-95 by casein kinase 2 (CK2) kinase augments cadherin 1 (CDH1)–mediated CBX3 degradation and RPA2 dynamics at stalled replication forks, which permits replication fork restart. Increased expression of CBX3 due to gene amplification or CK2 inhibitor treatment sensitizes prostate cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors while inducing replication stress and DNA damage. Our work reveals CBX3 as a key regulator of RPA2 function and DNA replication, suggesting that CBX3 could serve as an indicator for targeted therapy of cancer using PARP inhibitors.
Pharmacological inhibition of the ataxia telangiectasia and Rad3-related protein serine/threonine kinase (ATR; also known as FRAP-related protein (FRP1)) has emerged as a promising strategy for cancer treatment that exploits synthetic lethal interactions with proteins involved in DNA damage repair, overcomes resistance to other therapies and enhances antitumour immunity. Multiple novel, potent ATR inhibitors are being tested in clinical trials using biomarker-directed approaches and involving patients across a broad range of solid cancer types; some of these inhibitors have now entered phase III trials. Further insight into the complex interactions of ATR with other DNA replication stress response pathway components and with the immune system is necessary in order to optimally harness the potential of ATR inhibitors in the clinic and achieve hypomorphic targeting of the various ATR functions. Furthermore, a deeper understanding of the diverse range of predictive biomarkers of response to ATR inhibitors and of the intraclass differences between these agents could help to refine trial design and patient selection strategies. Key challenges that remain in the clinical development of ATR inhibitors include the optimization of their therapeutic index and the development of rational combinations with these agents. In this Review, we detail the molecular mechanisms regulated by ATR and their clinical relevance, and discuss the challenges that must be addressed to extend the benefit of ATR inhibitors to a broad population of patients with cancer.
DNA replication forks are subject to intricate surveillance and strict regulation by sophisticated cellular machinery. Such close regulation is necessary to ensure the accurate duplication of genetic information and to tackle the diverse endogenous and exogenous stresses that impede this process. Stalled replication forks are vulnerable to collapse, which is a major cause of genomic instability and carcinogenesis. Replication stress responses, which are organized via a series of coordinated molecular events, stabilize stalled replication forks and carry out fork reversal and restoration. DNA damage tolerance and repair pathways such as homologous recombination and Fanconi anemia also contribute to replication fork stabilization. The signaling network that mediates the transduction and interplay of these pathways is regulated by a series of post-translational modifications, including ubiquitination, which affects the activity, stability, and interactome of substrates. In particular, the ubiquitination of replication protein A and proliferating cell nuclear antigen at stalled replication forks promotes the recruitment of downstream regulators. In this review, we describe the ubiquitination-mediated signaling cascades that regulate replication fork progression and stabilization. In addition, we discuss the targeting of replication fork stability and ubiquitination system components as a potential therapeutic approach for the treatment of cancer.
