DOCSLIB.ORG

Phosphorylation by Mtorc1 Stablizes Skp2 and Regulates Its

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Phosphorylation by Mtorc1 Stablizes Skp2 and Regulates Its Geng et al. Molecular Cancer (2017) 16:83 DOI 10.1186/s12943-017-0649-0 RESEARCH Open Access Phosphorylation by mTORC1 stablizes Skp2 and regulates its oncogenic function in gastric cancer Qirong Geng1,2†, Jianjun Liu1,3†, Zhaohui Gong4†, Shangxiang Chen1,3†, Shuai Chen1, Xiaoxing Li1, Yue Lu1,2, Xiaofeng Zhu1, Hui-kuan Lin5 and Dazhi Xu1,3* Abstract Background: Both mTOR and Skp2 play critical roles in gastric cancer (GC) tumorigenesis. However, potential mechanisms for the association between these two proteins remains unidentified. Methods: The regulatory role for mTORC1 in Skp2 stability was tested using ubiquitination assay. The functions of p-Skp2 (phosphorylation of Skp2) were studied in vitro and in vivo. Expression of p-Skp2 and p-mTOR (phosphorylation of mTOR) were shown in GC lines and in 169 human primary GC tissues. Results: mTORC1 can directly interact with Skp2 and phosphorylated Skp2 at Ser64, which sequentially protect Skp2 from ubiquitination and degradation. Furthermore, the phospho-deficient p-Skp2 (S64) mutant significantly suppresses GC cell proliferation and tumorigenesis. The expression of p-Skp2 was associated with p-mTOR in GC cell lines and tissues. Interestingly, the combination of p-Skp2 and p-mTOR was a better predictor of survival than either factor alone. Conclusion: The mTORC1 function to regulate Skp2 by Ser64 phosphorylation may represent an oncogenic event in GC tumorigenesis. Moreover, our study also indicates that Skp2 Ser64 expression is a potential indicator in the treatment of GC patients using mTORC1 inhibitor. Keywords: mTORC1, Phosphorylation, Skp2, Gastric cancer Background of gastric cancer. For example, there is an increasing Gastric cancer (GC) is still an important public health interest in studying the role of the mammalian target of problem, with the third leading cause of cancer-related rapamycin (mTOR) in gastric cancer. We have also mortality worldwide [1]. The infection of Helicobacter revealed that mTOR is frequently activated in GC and phos- pylori and Epstein-Barr (EB) virus is thought to be asso- phorylated mTOR is associated with poor prognosis [7]. ciated with carcinogenesis of gastric cancer [2–6]. mTOR can form one of two complexes: mTOR com- Although D2 resection is the standard treatment for plex 1 (mTORC1) and mTOR complex 2 (mTORC2). gastric cancer, most of GC patients are diagnosed in a Activated mTORC1 phosphorylates two effector molecules, non-curable stage, especially in China. The understanding 4E-binding protein 1 (4EBP1) and S6 kinase 1 (S6K1), to of the molecular mechanisms is helpful to develop effect- stimulate protein synthesis supporting cell growth and cell ive treatment strategies. It has been known that several metabolism and angiogenesis [8–10]. signaling pathways play critical roles in the development As an oncoprotein, S-phase kinase associated protein 2 (Skp2) has also been reported activated in many types * Correspondence: [email protected] of cancers, included approximately 50% patients with †Equal contributors GC [11–13]. It is the key part of Skp-Cullin-F-box (SCF) 1 State Key Laboratory of Oncology in South China, Collaborative Innovation complex and participates in cell proliferation, metabolism Center for Cancer Medicine, Guangzhou, China 3Department of Gastric Surgery, Sun Yat-sen University Cancer Center, 651# East Dongfeng road, Guangzhou 510060, Guangdong Province, China Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Geng et al. Molecular Cancer (2017) 16:83 Page 2 of 12 and tumorigenesis by promoting ubiquitination of p27 treated with cycloheximide (100ug/ml) for 0 h to 5 h. and p21 [14–18]. Previously, we identified that macroh2A Then, the Skp2, mTOR and GAPDH were detected by is a new Skp2 SCF substrate, whose ubiquitination by immunoblotting. Skp2 inhibits the proliferation, colony formation and migration in breast cancer cells [19]. However, the Antibodies and reagents upstream regulation of Skp2 remains unclear [20]. Antibodies against Skp2, mTOR, phosphorylate- Recently, Jin et al. have found that Skp2 is a negative mTOR-ser2448, S6K, phosphorylate-S6K-ser389, 4EBP1, feedback regulator of amino-acid dependent mTORC1 phosphorylate-4EBP1-ser65 were purchased from Santa signaling and activation of mTORC1 signaling can re- Cruz Biotechnology Inc. Antibodies against FLAG, myc, cruit Skp2 to RagA [21]. This result indicates mTORC1 His were purchased from Sigma-Aldrich. Phosphorylate- activity can influence Skp2 activity and it is possible that Skp2-ser64 was generated by Zoonbio Biotechnology Skp2 phosphorylation mediated by mTORC1 [22]. Co.,Ltd. The mTOR inhibitor rapamycin were obtained Here we evaluated the mechanism by which mTORC1 from Sigma. controls Skp2 stability at the phosphorylation level. Our findings may help to understand the role of mTOR- dependent networks and their relationship to compensa- RNAi treatment tory pathways in GC tumorigenesis. All siRNAs were produced by GenePharma. According to the manufacturer’s protocol, 293 T cells were transfected Methods with siRNA oligonucleotides using the Lipofectamine Cell lines, stable cell lines and plasmids RNAiMAX transfection reagent (Invitrogen) for 48 h. Human GC cell lines (BGC 823, MKN 45, SGC 7901 The siRNAs for mTOR are as follows: and MGC 803) and 293 T cell line were purchased from mTOR no.1 sense:5’-GCAUCCAGCAGGAUAUCAA American Type Cell Culture (Manassas, VA). All cell TT-3’;antisense:5’-UUGAUAUCCUGCUGGAUGCTT-3’ lines were cultured in high-glucose RPIM-1640 or mTOR no.2 sense:5’- GCUGUCAGCCUGUCAGAAU DMEM supplemented with 10% fetal bovine serum TT -3’;antisense:5’- AUUCUGACAGGCUGACAGCTT-3’ (Gibco) in a moist chamber with 5% CO2 at 37 °C. Two cell lines (BGC 823 and MKN 45) were selected Immunoblotting and immunoprecipitation to generate stable cell lines in our research. The retro- Whole-cell extracts were lysed by lysis buffer (1 × Cell viral packaging system was purchased from Clontech. Lysis Buffer (Cell Signaling Technology) adding 1 mM According to the manufacture’s introduction, the recom- phenylmethylsulphonyl fluoride (PMSF) immediately binant retroviruses expressing the vector pcDNA 3.1 before use). Protein suspension liquid were boiled after and Skp2-wild-type (WT) or Skp2-S64A were generated. addition 6 × SDS sample buffer for 5–10 min at 100 °C. These retroviruses were infected to BGC 823 and MKN In immunoprecipitation experiment, whole-cell were lysed 45 and then were selected with 750ug/ml of G148 by E1A lysis buffer (50 mM HEPES (pH 7.5), 250 mM (Calbiochem). Mutation Skp2 was constructed by the NaCl, 5 mM EDTA, 0.1% NP-40, protease inhibitor cock- Quik Change Site-Directed Mutagenesis Kit (Stratagene), tail (Roche)). Immunoprecipitation was carried out either and then verified by DNA sequencing. Follow the by incubating appropriate antibody with cell lysate for manufacture’s introduction, the plasmids containing 2–3 h, followed by incubating Protein-A/G beads overnight pcDNA3.1-myc-mTOR, pcDNA3.1-FLAG-SKP2-wt and (Roche) or by incubating FLAG beads at 4 °C with lysate pcDNA3.1-FLAG-SKP2-S64A were constructed. overnight. Beeds were washed with ice-cold PBS three times, resuspended in SDS loading buffer. The protein Transfections, cell synchronization and cycloheximide samples were resolved by 10% SDS-PAGE and transfered chase assay to membrane (Sigma-aldrich). Then, the blots were Cell lines were transfected with variously plasmids with identified by various antibodies. lipofectamine 2000 (Life technologies) according to the manufacture’s introductions. Generally, cells seeds at 2.0 ×105 cells per well in a 6-well culture dish or at 1.0 × In vivo ubiquitination assay 106 cells per 10-cm dish were transfected with various In vivo ubiquitylation assays were performed as de- plasmids. For the synchronization experiment, 293 T cells scribed19. In brief, 293 T cells were transfected with the were treated with 2 mM thymidine for 12 h, and washed indicated plasmids for 24 h, and were treated with and released into fresh medium tissue culture dishs. As in- 20 μM LY, or 100 nM Wortmannin together with 20 μM dicated, transfection were performed during the last 4 h MG132 for 6 h prior to harvesting. The cell extracts before the second thymidine treatment. After the double- were then incubated with nickel beads for 3 h, washed, thymidine block, the 293 T cells were re-plated and and subjected to the Western blot analysis. Geng et al. Molecular Cancer (2017) 16:83 Page 3 of 12 Cell growth and in vivo tumorigenesis assay Similar results were also obtained by knockdown mTOR For cell growth assay, 5 × 103BGC823 stable cells in both BGC 823 (Fig. 1c) and MKN 45 cells (Additional with pcDNA, pcDNA-Skp2, or pcDNA-Skp2 S64A file 1: Figure S1B). Furthermore, the enhancement of and pcDNA-Skp2 S64D were seeded in 12-wells in mTORC1 activity by overexpression mTOR (Fig. 1d) or triplicate, harvested, and stained with trypan blue at insulin-like growth factor (IGF)-1 (Fig. 1e) in BGC 823 different days. Numbers of viable cells were directly cells leads to upregulation of Skp2 expression, suggesting counted under the microscope. The individual clone was that elevated mTOR activity upregulaties Skp2 in these picked and verified by Western blot analysis. two cell lines. These findings indicate that Skp2 is a For in vivo tumorigenesis assays, BGC823 stable cells candidate substrate of mTORC1. (1 × 106) mixed with matrigel (1:1) were injected sub- cutaneously into the left flank of 6-week-old nude mice.
Load more

Recommended publications
  • Cyclins, Cdks, E2f, Skp2, and More at the First International RB Tumor Suppressor Meeting
    Published OnlineFirst July 7, 2010; DOI: 10.1158/0008-5472.CAN-10-0358 Published OnlineFirst on July 7, 2010 as 10.1158/0008-5472.CAN-10-0358 Meeting Report Cancer Research Cyclins, Cdks, E2f, Skp2, and More at the First International RB Tumor Suppressor Meeting Rod Bremner1,3,4 and Eldad Zacksenhaus2,4,5,6 Abstract The RB1 gene was cloned because its inactivation causes the childhood ocular tumor, retinoblastoma. It is widely expressed, inactivated in most human malignancies, and present in diverse organisms from mammals to plants. Initially, retinoblastoma protein (pRB) was linked to cell cycle regulation, but it also regulates se- nescence, apoptosis, autophagy, differentiation, genome stability, immunity, telomere function, stem cell bi- ology, and embryonic development. In the 23 years since the gene was cloned, a formal international symposium focused on the RB pathway has not been held. The “First International RB Tumor Suppressor Meeting” (Toronto, Canada, November 19-21, 2009) established a biennial event to bring experts in the field together to discuss how the RB family (“pocket proteins”), as well as its regulators and effectors, influence biology and human disease. We summarize major new breakthroughs and emerging trends presented at the meeting. Cancer Res; 70(15); OF1–5. ©2010 AACR. Introduction critical for growth, thus their prominent expression could sensitize human cone precursors to RB1 mutation and un- Twenty speakers gave presentations on an astonishingly derlie a cone precursor origin of retinoblastoma (3). Studies diverse array of topics, underscoring the central role of reti- to date do not rule out an alternative possibility, that tumors noblastoma protein (pRB) in human biology (1).
    [Show full text]
  • The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression
    International Journal of Molecular Sciences Review The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression Tingting Zou and Zhenghong Lin * School of Life Sciences, Chongqing University, Chongqing 401331, China; [email protected] * Correspondence: [email protected] Abstract: The cell cycle is a collection of events by which cellular components such as genetic materials and cytoplasmic components are accurately divided into two daughter cells. The cell cycle transition is primarily driven by the activation of cyclin-dependent kinases (CDKs), which activities are regulated by the ubiquitin-mediated proteolysis of key regulators such as cyclins, CDK inhibitors (CKIs), other kinases and phosphatases. Thus, the ubiquitin-proteasome system (UPS) plays a pivotal role in the regulation of the cell cycle progression via recognition, interaction, and ubiquitination or deubiquitination of key proteins. The illegitimate degradation of tumor suppressor or abnormally high accumulation of oncoproteins often results in deregulation of cell proliferation, genomic instability, and cancer occurrence. In this review, we demonstrate the diversity and complexity of the regulation of UPS machinery of the cell cycle. A profound understanding of the ubiquitination machinery will provide new insights into the regulation of the cell cycle transition, cancer treatment, and the development of anti-cancer drugs. Keywords: cell cycle regulation; CDKs; cyclins; CKIs; UPS; E3 ubiquitin ligases; Deubiquitinases (DUBs) Citation: Zou, T.; Lin, Z. The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression. 1. Introduction Int. J. Mol. Sci. 2021, 22, 5754. https://doi.org/10.3390/ijms22115754 The cell cycle is a ubiquitous, complex, and highly regulated process that is involved in the sequential events during which a cell duplicates its genetic materials, grows, and di- Academic Editors: Kwang-Hyun Bae vides into two daughter cells.
    [Show full text]
  • Foxo3a Transcription Factor Is a Negative Regulator of Skp2 and Skp2 SCF Complex
    Oncogene (2013) 32, 78 --85 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Foxo3a transcription factor is a negative regulator of Skp2 and Skp2 SCF complex JWu1,2,4,5, S-W Lee2,3,5, X Zhang2,3, F Han2,3, S-Y Kwan2,3, X Yuan2, W-L Yang2,3, YS Jeong2,3, AH Rezaeian2, Y Gao2,3, Y-X Zeng1 and H-K Lin,2,3 Skp2 (S-phase kinase-associated protein-2) SCF complex displays E3 ligase activity and oncogenic activity by regulating protein ubiquitination and degradation, in turn regulating cell cycle entry, senescence and tumorigenesis. The maintenance of the integrity of Skp2 SCF complex is critical for its E3 ligase activity. The Skp2 F-box protein is a rate-limiting step and key factor in this complex, which binds to its protein substrates and triggers ubiquitination and degradation of its substrates. Skp2 is found to be overexpressed in numerous human cancers, which has an important role in tumorigenesis. The molecular mechanism by which the function of Skp2 and Skp2 SCF complex is regulated remains largely unknown. Here we show that Foxo3a transcription factor is a novel and negative regulator of Skp2 SCF complex. Foxo3a is found to be a transcriptional repressor of Skp2 gene expression by directly binding to the Skp2 promoter, thereby inhibiting Skp2 protein expression. Surprisingly, we found for the first time that Foxo3a also displays a transcription-independent activity by directly interacting with Skp2 and disrupting Skp2 SCF complex formation, in turn inhibiting Skp2 SCF E3 ligase activity and promoting p27 stability.
    [Show full text]
  • Coordinate Phosphorylation of Multiple Residues on Single AKT1 and AKT2 Molecules
    Oncogene (2014) 33, 3463–3472 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules H Guo1,6, M Gao1,6,YLu1, J Liang1, PL Lorenzi2, S Bai3, DH Hawke4,JLi1, T Dogruluk5, KL Scott5, E Jonasch3, GB Mills1 and Z Ding1 Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty- two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. Although basal AKT2 phosphorylation was sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K), basal AKT1 phosphorylation was essentially resistant.
    [Show full text]
  • Basal P21 Controls Population Heterogeneity in Cycling and Quiescent Cell Cycle States
    Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states K. Wesley Overtona, Sabrina L. Spencerb, William L. Noderera, Tobias Meyerb, and Clifford L. Wanga,1 Departments of aChemical Engineering and bChemical and Systems Biology, Stanford University, Stanford, CA 94305 Edited by Charles S. Peskin, New York University, Manhattan, NY, and approved August 27, 2014 (received for review May 27, 2014) Phenotypic heterogeneity within a population of genetically identical SCF/Skp2 then ubiquitinates p21, targeting it for proteasomal cells is emerging as a common theme in multiple biological systems, degradation (13). Thus, p21 both regulates and, through the ac- including human cell biology and cancer. Using live-cell imaging, flow tion of E3 ubiquitin ligase complexes that target p21, is regulated cytometry, and kinetic modeling, we showed that two states—quies- by active CDK2 bound to Cyclin E. cence and cell cycling—can coexist within an isogenic population of The p21–CDK2 control scheme is an example of a double- human cells and resulted from low basal expression levels of p21, a negative feedback loop. When stochastic gene expression leads Cyclin-dependent kinase (CDK) inhibitor (CKI). We attribute the p21- to fluctuations in factors involved in positive or double-negative dependent heterogeneity in cell cycle activity to double-negative feedback regulation, distinct cellular states within a population feedback regulation involving CDK2, p21, and E3 ubiquitin ligases. can arise (1, 14–18). Because of the role of p21 in the double- In support of this mechanism, analysis of cells at a point before cell negative feedback regulation of cell cycle activity, we hypothesized cycle entry (i.e., before the G1/S transition) revealed a p21–CDK2 axis that p21 controlled population heterogeneity in quiescent and that determines quiescent and cycling cell states.
    [Show full text]
  • Protein-Protein Interactions Among Signaling Pathways May Become New Therapeutic Targets in Liver Cancer (Review)
    ONCOLOGY REPORTS 35: 625-638, 2016 Protein-protein interactions among signaling pathways may become new therapeutic targets in liver cancer (Review) XIAO ZHANG1*, YULAN WANG1*, Jiayi WANG1,2 and FENYONG SUN1 1Department of Clinical Laboratory Medicine, Shanghai Tenth People's Hospital of Tongji University, Shanghai 200072; 2Translation Medicine of High Institute, Tongji University, Shanghai 200092, P.R. China Received May 29, 2015; Accepted July 6, 2015 DOI: 10.3892/or.2015.4464 Abstract. Numerous signaling pathways have been shown to be 1. Introduction dysregulated in liver cancer. In addition, some protein-protein interactions are prerequisite for the uncontrolled activation Liver cancer is the sixth most common cancer and the second or inhibition of these signaling pathways. For instance, in most common cause of cancer-associated mortality world- the PI3K/AKT signaling pathway, protein AKT binds with wide (1). Approximately 75% of all primary liver cancer types a number of proteins such as mTOR, FOXO1 and MDM2 to are hepatocellular carcinoma (HCC) that formed from liver play an oncogenic role in liver cancer. The aim of the present cells. Liver cancer can be formed from other structures in review was to focus on a series of important protein-protein the liver such as bile duct, blood vessels and immune cells. interactions that can serve as potential therapeutic targets Secondary liver cancer is a result of metastasis of cancer from in liver cancer among certain important pro-carcinogenic other body sites into the liver. The major cause of primary liver signaling pathways. The strategies of how to investigate and cancer is viral infection with either hepatitis C virus (HCV) analyze the protein-protein interactions are also included in or hepatitis B virus (HBV), which leads to massive inflamma- this review.
    [Show full text]
  • A Skp2 Autoinduction Loop and Restriction Point Control
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE JCB: REPORTprovided by PubMed Central A Skp2 autoinduction loop and restriction point control Yuval Yung,1 Janice L. Walker,1 James M. Roberts,2 and Richard K. Assoian1 1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 2Fred Hutchinson Cancer Research Center, Seattle, WA 98109 e describe a self-amplifying feedback loop continuous serum stimulation. Skp2 knock down inhibits that autoinduces Skp2 during G1 phase S phase entry in nontransformed mouse embryonic fi bro- progression. This loop, which contains Skp2 blasts but not in human papilloma virus–E7 expressing Wkip1 itself, p27 (p27), cyclin E–cyclin dependent kinase 2, fi broblasts. We propose that the essential role for Skp2- and the retinoblastoma protein, is closed through a newly dependent degradation of p27 is in the formation of an identifi ed, conserved E2F site in the Skp2 promoter. Inter- autoinduction loop that selectively controls the transition ference with the loop, by knockin of a Skp2-resistant p27 to mitogen-independence, and that Skp2-dependent pro- mutant (p27T187A), delays passage through the restriction teolysis may be dispensable when pocket proteins are point but does not interfere with S phase entry under constitutively inactivated. Introduction Skp2 is the limiting component of the E3 ligase controlling mitogen-independent cell cycle progression, also called the the proteosomal degradation of p27kip1 (p27) in late G1/S restriction point. phase (Carrano et al., 1999; Tsvetkov et al., 1999). Skp2 can also stimulate p27 degradation and S phase entry in serum- Results and discussion deprived cells (Sutterluty et al., 1999).
    [Show full text]
  • Short Article CAND1 Binds to Unneddylated CUL1 and Regulates
    Molecular Cell, Vol. 10, 1519–1526, December, 2002, Copyright 2002 by Cell Press CAND1 Binds to Unneddylated CUL1 Short Article and Regulates the Formation of SCF Ubiquitin E3 Ligase Complex Jianyu Zheng,1 Xiaoming Yang,1 Despite the importance of cullins in controlling many Jennifer M. Harrell,1 Sophia Ryzhikov,1 essential biological processes, the mechanism that reg- Eun-Hee Shim,1 Karin Lykke-Andersen,2 ulates the cullin-containing ubiquitin E3 ligases remains Ning Wei,2 Hong Sun,1 Ryuji Kobayashi,3 unclear. In SCF, the F box proteins are short-lived pro- and Hui Zhang1,4 teins that undergo CUL1/SKP1-dependent degradation 1Department of Genetics (Wirbelauer et al., 2000; Zhou and Howley, 1998). Dele- Yale University School of Medicine tion of the F box region abolishes the binding of F box 333 Cedar Street proteins to SKP1 and CUL1, and consequently increases 2 Department of Molecular, Cellular, the stability of F box proteins. This substrate-indepen- and Developmental Biology dent proteolysis of F box proteins is likely the result of Yale University autoubiquitination by the ubiquitin E2 and E1 enzymes New Haven, Connecticut 06520 through a CUL1/SKP1-dependent mechanism. 3 Cold Spring Harbor Laboratory The carboxy-terminal ends of cullins are often covalently Cold Spring Harbor, New York 11724 modified by a ubiquitin-like protein, NEDD8/RUB1, and this modification appears to associate with active E3 li- gases (Hochstrasser, 2000). Like ubiquitin modification, Summary neddylation requires E1 (APP-BP1 and UBA3)-activating and E2 (UBC12)-conjugating enzymes (Hochstrasser, The SCF ubiquitin E3 ligase regulates ubiquitin-depen- 2000).
    [Show full text]
  • Skp2 in the Ubiquitin‐Proteasome System: a Comprehensive Review
    Received: 19 November 2019 | Revised: 26 March 2020 | Accepted: 27 April 2020 DOI: 10.1002/med.21675 REVIEW ARTICLE Skp2 in the ubiquitin‐proteasome system: A comprehensive review Moges Dessale Asmamaw | Ying Liu | Yi-Chao Zheng | Xiao‐Jing Shi | Hong‐Min Liu State Key Laboratory of Esophageal Cancer Prevention & Treatment, Key Laboratory of Abstract Advanced Drug Preparation Technologies, The ubiquitin‐proteasome system (UPS) is a complex Henan Key Laboratory of Drug Quality Control & Evaluation, School of Pharmaceutical process that regulates protein stability and activity by Sciences, Zhengzhou University, Ministry of the sequential actions of E1, E2 and E3 enzymes to in- Education of China, Zhengzhou, Henan, China fluence diverse aspects of eukaryotic cells. However, due Correspondence to the diversity of proteins in cells, substrate selection is Xiao‐Jing Shi and Hong‐Min Liu, State Key Laboratory of Esophageal Cancer Prevention & a highly critical part of the process. As a key player in Treatment; Key Laboratory of Advanced Drug UPS, E3 ubiquitin ligases recruit substrates for ubiquiti- Preparation Technologies, Ministry of Education of China; Henan Key Laboratory of Drug nation specifically. Among them, RING E3 ubiquitin li- Quality Control & Evaluation; School of gases which are the most abundant E3 ubiquitin ligases Pharmaceutical Sciences, Zhengzhou University, 450001 Zhengzhou, Henan, China. contribute to diverse cellular processes. The multisubunit Email: [email protected] (X‐JS) and cullin‐RING ligases (CRLs) are the largest family of RING [email protected] (H‐ML) E3 ubiquitin ligases with tremendous plasticity in sub- Funding information strate specificity and regulate a vast array of cellular National Key Research Program of Proteins, functions.
    [Show full text]
  • A Haploid Genetic Screen Identifies the G1/S Regulatory Machinery As a Determinant of Wee1 Inhibitor Sensitivity
    A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity Anne Margriet Heijinka, Vincent A. Blomenb, Xavier Bisteauc, Fabian Degenera, Felipe Yu Matsushitaa, Philipp Kaldisc,d, Floris Foijere, and Marcel A. T. M. van Vugta,1 aDepartment of Medical Oncology, University Medical Center Groningen, University of Groningen, 9723 GZ Groningen, The Netherlands; bDivision of Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; cInstitute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos#3-09, Singapore 138673, Republic of Singapore; dDepartment of Biochemistry, National University of Singapore, Singapore 117597, Republic of Singapore; and eEuropean Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands Edited by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved October 21, 2015 (received for review March 17, 2015) The Wee1 cell cycle checkpoint kinase prevents premature mitotic Wee1 kinase at tyrosine (Tyr)-15 to prevent unscheduled Cdk1 entry by inhibiting cyclin-dependent kinases. Chemical inhibitors activity (5, 6). Conversely, timely activation of Cdk1 depends on of Wee1 are currently being tested clinically as targeted anticancer Tyr-15 dephosphorylation by one of the Cdc25 phosphatases drugs. Wee1 inhibition is thought to be preferentially cytotoxic in (7–10). When DNA is damaged, the downstream DNA damage p53-defective cancer cells. However, TP53 mutant cancers do not response (DDR) kinases Chk1 and Chk2 inhibit Cdc25 phos- respond consistently to Wee1 inhibitor treatment, indicating the phatases through direct phosphorylation, which blocks Cdk1 existence of genetic determinants of Wee1 inhibitor sensitivity other activation (11–13).
    [Show full text]
  • Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in P27kip1 Degradation
    Discriminative SKP2 Interactions with CDK- Cyclin Complexes Support a Cyclin A- Specific Role in p27KIP1 Degradation. Item Type Article Authors Salamina, Marco; Montefiore, Bailey C; Liu, Mengxi; Wood, Daniel J; Heath, Richard; Ault, James R; Wang, Lan-Zhen; Korolchuk, Svitlana; Baslé, Arnaud; Pastok, Martyna W; Reeks, Judith; Tatum, Natalie J; Sobott, Frank; Arold, Stefan T.; Pagano, Michele; Noble, Martin E M; Endicott, Jane A Citation Salamina, M., Montefiore, B. C., Liu, M., Wood, D. J., Heath, R., Ault, J. R., … Endicott, J. A. (2021). Discriminative SKP2 interactions with CDK-cyclin complexes support a cyclin A- specific role in p27KIP1 degradation. Journal of Molecular Biology, 166795. doi:10.1016/j.jmb.2020.166795 Eprint version Publisher's Version/PDF DOI 10.1016/j.jmb.2020.166795 Publisher Elsevier BV Journal Journal of molecular biology Rights This is an open access article under the CC BY license. Download date 29/09/2021 07:35:00 Item License http://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/10754/667206 Research Article Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation Marco Salamina 1, Bailey C. Montefiore 1, Mengxi Liu 2, Daniel J. Wood 1, Richard Heath 1, James R. Ault 3, Lan-Zhen Wang 1, Svitlana Korolchuk 1, Arnaud Basle´ 4, Martyna W. Pastok 1, Judith Reeks 1, Natalie J. Tatum 1, ⇑ Frank Sobott 3, Stefan T. Arold 5,6, Michele Pagano 2, Martin E. M. Noble 1 and ⇑ Jane A. Endicott 1 1 - Newcastle University Centre for Cancer,
    [Show full text]
  • The Roles of Cyclin-Dependent Kinases in Cell-Cycle Progression and Therapeutic Strategies in Human Breast Cancer
    International Journal of Molecular Sciences Review The Roles of Cyclin-Dependent Kinases in Cell-Cycle Progression and Therapeutic Strategies in Human Breast Cancer 1,2, 1,2, 1,2, 1,2 1,2 Lei Ding y, Jiaqi Cao y, Wen Lin y, Hongjian Chen , Xianhui Xiong , Hongshun Ao 1,2, Min Yu 1,2, Jie Lin 1,2 and Qinghua Cui 1,2,* 1 Lab of Biochemistry & Molecular Biology, School of Life Sciences, Yunnan University, Kunming 650091, China; [email protected] (L.D.); [email protected] (J.C.); [email protected] (W.L.); [email protected] (H.C.); [email protected] (X.X.); [email protected] (H.A.); [email protected] (M.Y.); [email protected] (J.L.) 2 Key Lab of Molecular Cancer Biology, Yunnan Education Department, Kunming 650091, China * Correspondence: [email protected] The authors contributed equally to this work. y Received: 31 December 2019; Accepted: 24 February 2020; Published: 13 March 2020 Abstract: Cyclin-dependent kinases (CDKs) are serine/threonine kinases whose catalytic activities are regulated by interactions with cyclins and CDK inhibitors (CKIs). CDKs are key regulatory enzymes involved in cell proliferation through regulating cell-cycle checkpoints and transcriptional events in response to extracellular and intracellular signals. Not surprisingly, the dysregulation of CDKs is a hallmark of cancers, and inhibition of specific members is considered an attractive target in cancer therapy. In breast cancer (BC), dual CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, combined with other agents, were approved by the Food and Drug Administration (FDA) recently for the treatment of hormone receptor positive (HR+) advanced or metastatic breast cancer (A/MBC), as well as other sub-types of breast cancer.
    [Show full text]