The Protein Journal

, Volume 37, Issue 2, pp 132–143 | Cite as

The Catalytically Inactive Mutation of the Ubiquitin-Conjugating Enzyme CDC34 Affects its Stability and Cell Proliferation

  • Xun Liu
  • Yang Zhang
  • Zhanhong Hu
  • Qian Li
  • Lu Yang
  • Guoqiang Xu
Article

Abstract

The ubiquitin proteasome system (UPS) plays important roles in the regulation of protein stability, localization, and activity. A myriad of studies have focused on the functions of ubiquitin ligases E3s and deubiquitinating enzymes DUBs due to their specificity in the recognition of downstream substrates. However, the roles of the most ubiquitin-conjugating enzymes E2s are not completely understood except that they transport the activated ubiquitin and form E2–E3 protein complexes. Ubiquitin-conjugating enzyme CDC34 can promote the degradation of downstream targets through the UPS whereas its non-catalytic functions are still elusive. Here, we find that mutation of the catalytically active cysteine to serine (C93S) results in the reduced ubiquitination, increased stability, and attenuated degradation rate of CDC34. Through semi-quantitative proteomics, we identify the CDC34-interacting proteins and discover that the wild-type and mutant proteins have many differentially interacted proteins. Detailed examination finds that some of them are involved in the regulation of gene expression, cell growth, and cell proliferation. Cell proliferation assay reveals that both the wild-type and C93S proteins affect the proliferation of a cancer cell line. Database analyses show that CDC34 mRNA is highly expressed in multiple cancers, which is correlated with the reduced patient survival rate. This work may help to elucidate the enzymatic and non-enzymatic functions of this protein and might provide additional insights for drug discovery targeting E2s.

Keywords

Catalytic site CDC34 Cell proliferation Protein degradation Proteomics Ubiquitination 

Abbreviations

CCK-8

Cell counting kit-8

CDC34

Ubiquitin-conjugating enzyme E2 R1 or cell division cycle 34

CHX

Cycloheximide

CSNK1G2

Casein kinase I isoform γ2

GNL3

Guanine nucleotide-binding protein-like 3

HEK

Human embryonic kidney

MCM7

DNA replication licensing factor MCM7

MS

Mass spectrometry

NC

Negative control

PCR

Polymerase chain reaction

PEI

Polyethyleneimine

PSM

Peptide spectrum match

SCF

Skp-cullin-1-F-box

SDS–PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SEM

Standard error of measurements

Ub

Ubiquitin

UPS

Ubiquitin proteasome system

C93S

C93S mutant

WT

CDC34 wild-type

Notes

Acknowledgements

We thank Yarong Wang at the Mass Spectrometry core facility of the Medical School of Soochow University for the assistance during the MS analysis. We also appreciate Dr. Xiaoyan Qiu at Soochow University for her critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (31670833 & 31700722), Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX17_2040), Jiangsu Key Laboratory of Neuropsychiatric Diseases (BM2013003), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human Participants and Animals

This article does not contain any studies with human participants or animals performed by any of the authors

Supplementary material

10930_2018_9766_MOESM1_ESM.docx (2.5 mb)
Supplementary material 1 (DOCX 2541 KB)
10930_2018_9766_MOESM2_ESM.xlsx (47 kb)
Supplementary material 2 (XLSX 46 KB)

References

  1. 1.
    Hershko A, Ciechanover A (1992) The ubiquitin system for protein degradation. Annu Rev Biochem 61:761–807CrossRefGoogle Scholar
  2. 2.
    Haas AL, Rose IA (1982) The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis. J Biol Chem 257:10329–10337Google Scholar
  3. 3.
    Haas AL, Warms JV, Hershko A, Rose IA (1982) Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation. J Biol Chem 257:2543–2548Google Scholar
  4. 4.
    Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–533CrossRefGoogle Scholar
  5. 5.
    Ardley HC, Robinson PA (2005) E3 ubiquitin ligases. Essays Biochem 41:15–30CrossRefGoogle Scholar
  6. 6.
    Stewart MD, Ritterhoff T, Klevit RE, Brzovic PS (2016) E2 enzymes: more than just middle men. Cell Res 26:423–440CrossRefGoogle Scholar
  7. 7.
    Ye Y, Rape M (2009) Building ubiquitin chains: E2 enzymes at work. Nat Rev Mol Cell Biol 10:755–764CrossRefGoogle Scholar
  8. 8.
    van Wijk SJ, Timmers HT (2010) The family of ubiquitin-conjugating enzymes (E2s): deciding between life and death of proteins. FASEB J 24:981–993CrossRefGoogle Scholar
  9. 9.
    Sandoval D, Hill S, Ziemba A, Lewis S, Kuhlman B, Kleiger G (2015) Ubiquitin-conjugating enzyme Cdc34 and ubiquitin ligase Skp1-cullin-F-box ligase (SCF) interact through multiple conformations. J Biol Chem 290:1106–1118CrossRefGoogle Scholar
  10. 10.
    Petroski MD, Deshaies RJ (2005) Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34. Cell 123:1107–1120CrossRefGoogle Scholar
  11. 11.
    Kleiger G, Saha A, Lewis S, Kuhlman B, Deshaies RJ (2009) Rapid E2-E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. Cell 139:957–968CrossRefGoogle Scholar
  12. 12.
    Suryadinata R, Holien JK, Yang G, Parker MW, Papaleo E, Sarcevic B (2013) Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34. Cell Cycle 12:1732–1744CrossRefGoogle Scholar
  13. 13.
    Charrasse S, Carena I, Brondani V, Klempnauer KH, Ferrari S (2000) Degradation of B-Myb by ubiquitin-mediated proteolysis: involvement of the Cdc34-SCF(p45Skp2) pathway. Oncogene 19:2986–2995CrossRefGoogle Scholar
  14. 14.
    Gonen H, Bercovich B, Orian A, Carrano A, Takizawa C, Yamanaka K, Pagano M, Iwai K, Ciechanover A (1999) Identification of the ubiquitin carrier proteins, E2s, involved in signal-induced conjugation and subsequent degradation of IκBα. J Biol Chem 274:14823–14830CrossRefGoogle Scholar
  15. 15.
    Butz N, Ruetz S, Natt F, Hall J, Weiler J, Mestan J, Ducarre M, Grossenbacher R, Hauser P, Kempf D, Hofmann F (2005) The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27Kip1 protein levels. Exp Cell Res 303:482–493CrossRefGoogle Scholar
  16. 16.
    Kaiser P, Sia RA, Bardes EG, Lew DJ, Reed SI (1998) Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1. Genes Dev 12:2587–2597CrossRefGoogle Scholar
  17. 17.
    Michael WM, Newport J (1998) Coupling of mitosis to the completion of S phase through Cdc34-mediated degradation of Wee1. Science 282:1886–1889CrossRefGoogle Scholar
  18. 18.
    Eliseeva E, Pati D, Diccinanni MB, Yu AL, Mohsin SK, Margolin JF, Plon SE (2001) Expression and localization of the CDC34 ubiquitin-conjugating enzyme in pediatric acute lymphoblastic leukemia. Cell Growth Differ 12:427–433Google Scholar
  19. 19.
    Chauhan D, Li G, Hideshima T, Podar K, Shringarpure R, Mitsiades C, Munshi N, Yew PR, Anderson KC (2004) Blockade of ubiquitin-conjugating enzyme CDC34 enhances anti-myeloma activity of Bortezomib/Proteasome inhibitor PS-341. Oncogene 23:3597–3602CrossRefGoogle Scholar
  20. 20.
    Tanaka K, Kondoh N, Shuda M, Matsubara O, Imazeki N, Ryo A, Wakatsuki T, Hada A, Goseki N, Igari T, Hatsuse K, Aihara T, Horiuchi S, Yamamoto N, Yamamoto M (2001) Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas. Biochim Biophys Acta 1536:1–12CrossRefGoogle Scholar
  21. 21.
    Ceccarelli DF, Tang X, Pelletier B, Orlicky S, Xie W, Plantevin V, Neculai D, Chou YC, Ogunjimi A, Al-Hakim A, Varelas X, Koszela J, Wasney GA, Vedadi M, Dhe-Paganon S, Cox S, Xu S, Lopez-Girona A, Mercurio F, Wrana J, Durocher D, Meloche S, Webb DR, Tyers M, Sicheri F (2011) An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme. Cell 145:1075–1087CrossRefGoogle Scholar
  22. 22.
    Arrigoni A, Bertini L, De Gioia L, Papaleo E (2014) Inhibitors of the Cdc34 acidic loop: a computational investigation integrating molecular dynamics, virtual screening and docking approaches. FEBS Open Bio 4:473–484CrossRefGoogle Scholar
  23. 23.
    Xu G, Jiang X, Jaffrey SR (2013) A mental retardation-linked nonsense mutation in cereblon is rescued by proteasome inhibition. J Biol Chem 288:29573–29585CrossRefGoogle Scholar
  24. 24.
    Hou XO, Si JM, Ren HG, Chen D, Wang HF, Ying Z, Hu QS, Gao F, Wang GH (2015) Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells. Acta Pharmacol Sin 36:1300–1307CrossRefGoogle Scholar
  25. 25.
    Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21:3666–3672CrossRefGoogle Scholar
  26. 26.
    Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM (1999) Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20:601–605CrossRefGoogle Scholar
  27. 27.
    Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860CrossRefGoogle Scholar
  28. 28.
    Xu G, Deglincerti A, Paige JS, Jaffrey SR (2014) Profiling lysine ubiquitination by selective enrichment of ubiquitin remnant-containing peptides. Methods Mol Biol 1174:57–71CrossRefGoogle Scholar
  29. 29.
    Duan W, Chen S, Zhang Y, Li D, Wang R, Chen S, Li J, Qiu X, Xu G (2016) Protein C-terminal enzymatic labeling identifies novel caspase cleavages during the apoptosis of multiple myeloma cells induced by kinase inhibition. Proteomics 16:60–69CrossRefGoogle Scholar
  30. 30.
    The UniProt Consortium (2015) UniProt: a hub for protein information. Nucleic Acids Res 43:D204-212CrossRefGoogle Scholar
  31. 31.
    Elias JE, Gygi SP (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4:207–214CrossRefGoogle Scholar
  32. 32.
    Liu H, Sadygov RG, Yates JR 3rd (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76:4193–4201CrossRefGoogle Scholar
  33. 33.
    de Bie P, Ciechanover A (2011) Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms. Cell Death Differ 18:1393–1402CrossRefGoogle Scholar
  34. 34.
    Block K, Boyer TG, Yew PR (2001) Phosphorylation of the human ubiquitin-conjugating enzyme, CDC34, by casein kinase 2. J Biol Chem 276:41049–41058CrossRefGoogle Scholar
  35. 35.
    Wu K, Chong RA, Yu Q, Bai J, Spratt DE, Ching K, Lee C, Miao H, Tappin I, Hurwitz J, Zheng N, Shaw GS, Sun Y, Felsenfeld DP, Sanchez R, Zheng JN, Pan ZQ (2016) Suramin inhibits cullin-RING E3 ubiquitin ligases. Proc Natl Acad Sci USA 113:E2011-2018Google Scholar
  36. 36.
    Gnjatic S, Cao Y, Reichelt U, Yekebas EF, Nolker C, Marx AH, Erbersdobler A, Nishikawa H, Hildebrandt Y, Bartels K, Horn C, Stahl T, Gout I, Filonenko V, Ling KL, Cerundolo V, Luetkens T, Ritter G, Friedrichs K, Leuwer R, Hegewisch-Becker S, Izbicki JR, Bokemeyer C, Old LJ, Atanackovic D (2010) NY-CO-58/KIF2C is overexpressed in a variety of solid tumors and induces frequent T cell responses in patients with colorectal cancer. Int J Cancer 127:381–393Google Scholar
  37. 37.
    Li Y, Lu W, Chen D, Boohaker RJ, Zhai L, Padmalayam I, Wennerberg K, Xu B, Zhang W (2015) KIFC1 is a novel potential therapeutic target for breast cancer. Cancer Biol Ther 16:1316–1322CrossRefGoogle Scholar
  38. 38.
    Rhodes DR, Kalyana-Sundaram S, Mahavisno V, Varambally R, Yu J, Briggs BB, Barrette TR, Anstet MJ, Kincead-Beal C, Kulkarni P, Varambally S, Ghosh D, Chinnaiyan AM (2007) Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer gene expression profiles. Neoplasia 9:166–180CrossRefGoogle Scholar
  39. 39.
    Szasz AM, Lanczky A, Nagy A, Forster S, Hark K, Green JE, Boussioutas A, Busuttil R, Szabo A, Gyorffy B (2016) Cross-validation of survival associated biomarkers in gastric cancer using transcriptomic data of 1065 patients. Oncotarget 7:49322–49333CrossRefGoogle Scholar
  40. 40.
    Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E (2015) PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res 43:D512-520CrossRefGoogle Scholar
  41. 41.
    Scaglione KM, Bansal PK, Deffenbaugh AE, Kiss A, Moore JM, Korolev S, Cocklin R, Goebl M, Kitagawa K, Skowyra D (2007) SCF E3-mediated autoubiquitination negatively regulates activity of Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo. Mol Cell Biol 27:5860–5870CrossRefGoogle Scholar
  42. 42.
    Scortegagna M, Subtil T, Qi J, Kim H, Zhao W, Gu W, Kluger H, Ronai ZA (2011) USP13 enzyme regulates Siah2 ligase stability and activity via noncatalytic ubiquitin-binding domains. J Biol Chem 286:27333–27341CrossRefGoogle Scholar
  43. 43.
    Skaug B, Chen J, Du F, He J, Ma A, Chen ZJ (2011) Direct, noncatalytic mechanism of IKK inhibition by A20. Mol Cell 44:559–571CrossRefGoogle Scholar
  44. 44.
    Lei M (2005) The MCM complex: its role in DNA replication and implications for cancer therapy. Curr Cancer Drug Targets 5:365–380CrossRefGoogle Scholar
  45. 45.
    Kang W, Tong JH, Chan AW, Cheng AS, Yu J, To K (2014) MCM7 serves as a prognostic marker in diffuse-type gastric adenocarcinoma and siRNA-mediated knockdown suppresses its oncogenic function. Oncol Rep 31:2071–2078CrossRefGoogle Scholar
  46. 46.
    Chen J, Dong S, Hu J, Duan B, Yao J, Zhang R, Zhou H, Sheng H, Gao H, Li S, Zhang X (2015) Guanine nucleotide binding protein-like 3 is a potential prognosis indicator of gastric cancer. Int J Clin Exp Pathol 8:13273–13278Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric DiseasesSoochow UniversitySuzhouChina

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