Epstein-Barr Virus Nuclear Antigen 3C Facilitates G1-S Transition by Stabilizing and Enhancing the Function of Cyclin D1
EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130–160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1–50), and C-terminal domain (residues 171–240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.
Vyšlo v časopise:
Epstein-Barr Virus Nuclear Antigen 3C Facilitates G1-S Transition by Stabilizing and Enhancing the Function of Cyclin D1. PLoS Pathog 7(2): e32767. doi:10.1371/journal.ppat.1001275
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1001275
Souhrn
EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130–160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1–50), and C-terminal domain (residues 171–240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.
Zdroje
1. RobertsonES
2010
Epstein-Barr virus: latency and transformation.
Norwich, United Kingdom
Caister Academic Press
2. KieffE
RickinsonAB
2002
Epstein-Barr virus and its replication.
KnipeD
HowleyP
Fields virology, 4th ed
Philadelphia
Lippincott Williams & Wilkins
2511
2573
3. O'NionsJ
AlldayMJ
2004
Deregulation of the cell cycle by the Epstein-Barr virus.
Adv Cancer Res
92
119
186
4. BajajBG
MurakamiM
CaiQ
VermaSC
LanK
2008
Epstein-Barr virus nuclear antigen 3C interacts with and enhances the stability of the c-Myc oncoprotein.
J Virol
82
4082
4090
5. BainM
WatsonRJ
FarrellPJ
AlldayMJ
1996
Epstein-Barr virus nuclear antigen 3C is a powerful repressor of transcription when tethered to DNA.
J Virol
70
2481
2489
6. RosendorffA
IllanesD
DavidG
LinJ
KieffE
2004
EBNA3C coactivation with EBNA2 requires a SUMO homology domain.
J Virol
78
367
377
7. CotterMA2nd
RobertsonES
2000
Modulation of histone acetyltransferase activity through interaction of epstein-barr nuclear antigen 3C with prothymosin alpha.
Mol Cell Biol
20
5722
5735
8. KnightJS
LanK
SubramanianC
RobertsonES
2003
Epstein-Barr virus nuclear antigen 3C recruits histone deacetylase activity and associates with the corepressors mSin3A and NCoR in human B-cell lines.
J Virol
77
4261
4272
9. TouitouR
HickabottomM
ParkerG
CrookT
AlldayMJ
2001
Physical and functional interactions between the corepressor CtBP and the Epstein-Barr virus nuclear antigen EBNA3C.
J Virol
75
7749
7755
10. SubramanianC
CotterMA2nd
RobertsonES
2001
Epstein-Barr virus nuclear protein EBNA-3C interacts with the human metastatic suppressor Nm23-H1: a molecular link to cancer metastasis.
Nat Med
7
350
355
11. YanX
MouilletJF
OuQ
SadovskyY
2003
A novel domain within the DEAD-box protein DP103 is essential for transcriptional repression and helicase activity.
Mol Cell Biol
23
414
423
12. KnightJS
SharmaN
RobertsonES
2005
SCFSkp2 complex targeted by Epstein-Barr virus essential nuclear antigen.
Mol Cell Biol
25
1749
1763
13. SubramanianC
HasanS
RoweM
HottigerM
OrreR
2002
Epstein-Barr virus nuclear antigen 3C and prothymosin alpha interact with the p300 transcriptional coactivator at the CH1 and CH3/HAT domains and cooperate in regulation of transcription and histone acetylation.
J Virol
76
4699
4708
14. YiF
SahaA
MurakamiM
KumarP
KnightJS
2009
Epstein-Barr virus nuclear antigen 3C targets p53 and modulates its transcriptional and apoptotic activities.
Virology
388
236
247
15. KnightJS
RobertsonES
2004
Epstein-Barr virus nuclear antigen 3C regulates cyclin A/p27 complexes and enhances cyclin A-dependent kinase activity.
J Virol
78
1981
1991
16. KnightJS
SharmaN
KalmanDE
RobertsonES
2004
A cyclin-binding motif within the amino-terminal homology domain of EBNA3C binds cyclin A and modulates cyclin A-dependent kinase activity in Epstein-Barr virus-infected cells.
J Virol
78
12857
12867
17. KnightJS
SharmaN
RobertsonES
2005
Epstein-Barr virus latent antigen 3C can mediate the degradation of the retinoblastoma protein through an SCF cellular ubiquitin ligase.
Proc Natl Acad Sci U S A
102
18562
18566
18. SahaA
MurakamiM
KumarP
BajajB
SimsK
2009
Epstein-Barr virus nuclear antigen 3C augments Mdm2-mediated p53 ubiquitination and degradation by deubiquitinating Mdm2.
J Virol
83
4652
4669
19. ChoudhuriT
VermaSC
LanK
MurakamiM
RobertsonES
2007
The ATM/ATR signaling effector Chk2 is targeted by Epstein-Barr virus nuclear antigen 3C to release the G2/M cell cycle block.
J Virol
81
6718
6730
20. ParkerGA
CrookT
BainM
SaraEA
FarrellPJ
1996
Epstein-Barr virus nuclear antigen (EBNA)3C is an immortalizing oncoprotein with similar properties to adenovirus E1A and papillomavirus E7.
Oncogene
13
2541
2549
21. ParkerGA
TouitouR
AlldayMJ
2000
Epstein-Barr virus EBNA3C can disrupt multiple cell cycle checkpoints and induce nuclear division divorced from cytokinesis.
Oncogene
19
700
709
22. BloomJ
CrossFR
2007
Multiple levels of cyclin specificity in cell-cycle control.
Nat Rev Mol Cell Biol
8
149
160
23. BaldinV
LukasJ
MarcoteMJ
PaganoM
DraettaG
1993
Cyclin D1 is a nuclear protein required for cell cycle progression in G1.
Genes Dev
7
812
821
24. BenzenoS
LuF
GuoM
BarbashO
ZhangF
2006
Identification of mutations that disrupt phosphorylation-dependent nuclear export of cyclin D1.
Oncogene
25
6291
6303
25. GansaugeS
GansaugeF
RamadaniM
StobbeH
RauB
1997
Overexpression of cyclin D1 in human pancreatic carcinoma is associated with poor prognosis.
Cancer Res
57
1634
1637
26. LinDI
BarbashO
KumarKG
WeberJD
HarperJW
2006
Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex.
Mol Cell
24
355
366
27. DiehlJA
ChengM
RousselMF
SherrCJ
1998
Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization.
Genes Dev
12
3499
3511
28. CampoE
RaffeldM
JaffeES
1999
Mantle-cell lymphoma.
Semin Hematol
36
115
127
29. YatabeY
NakamuraS
SetoM
KurodaH
KagamiY
1996
Clinicopathologic study of PRAD1/cyclin D1 overexpressing lymphoma with special reference to mantle cell lymphoma. A distinct molecular pathologic entity.
Am J Surg Pathol
20
1110
1122
30. NakagawaH
WangTC
ZukerbergL
OdzeR
TogawaK
1997
The targeting of the cyclin D1 oncogene by an Epstein-Barr virus promoter in transgenic mice causes dysplasia in the tongue, esophagus and forestomach.
Oncogene
14
1185
1190
31. KimJK
DiehlJA
2009
Nuclear cyclin D1: an oncogenic driver in human cancer.
J Cell Physiol
220
292
296
32. PalmeroI
HolderA
SinclairAJ
DicksonC
PetersG
1993
Cyclins D1 and D2 are differentially expressed in human B-lymphoid cell lines.
Oncogene
8
1049
1054
33. MeyyappanM
WongH
HullC
RiabowolKT
1998
Increased expression of cyclin D2 during multiple states of growth arrest in primary and established cells.
Mol Cell Biol
18
3163
3172
34. SinclairAJ
PalmeroI
PetersG
FarrellPJ
1994
EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalization of resting human B lymphocytes by Epstein-Barr virus.
EMBO J
13
3321
3328
35. KimHR
JeongJA
ParkCH
LeeSK
LeeWK
2002
A role for cell cycle proteins in the serum-starvation resistance of Epstein-Barr virus immortalized B lymphocytes.
Biochem Cell Biol
80
407
413
36. ParkCH
KimHR
KimJ
JangSH
LeeKY
2004
Latent membrane protein 1 of Epstein-Barr virus plays an important role in the serum starvation resistance of Epstein-Barr virus-immortalized B lymphocytes.
J Cell Biochem
91
777
785
37. MuraiY
DobashiY
OkadaE
IshizawaS
ShiotaM
2001
Study on the role of G1 cyclins in Epstein-Barr virus-associated human lymphomas maintained in severe combined immune deficiency (SCID) mice.
Int J Cancer
92
232
239
38. LuJ
VermaSC
MurakamiM
CaiQ
KumarP
2009
Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus (KSHV) upregulates survivin expression in KSHV-Associated B-lymphoma cells and contributes to their proliferation.
J Virol
83
7129
7141
39. PontenJ
SakselaE
1967
Two established in vitro cell lines from human mesenchymal tumours.
Int J Cancer
2
434
447
40. HalderS
MurakamiM
VermaSC
KumarP
YiF
2009
Early events associated with infection of Epstein-Barr virus infection of primary B-cells.
PLoS One
4
e7214
41. LaemmliUK
1970
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
680
685
42. KlierM
AnastasovN
HermannA
MeindlT
AngermeierD
2008
Specific lentiviral shRNA-mediated knockdown of cyclin D1 in mantle cell lymphoma has minimal effects on cell survival and reveals a regulatory circuit with cyclin D2.
Leukemia
22
2097
2105
43. O'NionsJ
AlldayMJ
2003
Epstein-Barr virus can inhibit genotoxin-induced G1 arrest downstream of p53 by preventing the inactivation of CDK2.
Oncogene
22
7181
7191
44. MaruoS
WuY
IshikawaS
KandaT
IwakiriD
2006
Epstein-Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells.
Proc Natl Acad Sci U S A
103
19500
19505
45. ArvanitakisL
YaseenN
SharmaS
1995
Latent membrane protein-1 induces cyclin D2 expression, pRb hyperphosphorylation, and loss of TGF-beta 1-mediated growth inhibition in EBV-positive B cells.
J Immunol
155
1047
1056
46. SiegertJL
RushtonJJ
SellersWR
KaelinWGJr
RobbinsPD
2000
Cyclin D1 suppresses retinoblastoma protein-mediated inhibition of TAFII250 kinase activity.
Oncogene
19
5703
5711
47. ZwickerJ
BrusselbachS
JoossKU
SewingA
BehnM
1999
Functional domains in cyclin D1: pRb-kinase activity is not essential for transformation.
Oncogene
18
19
25
48. ZhouY
LiJ
XuK
HuSX
BenedictWF
1994
Further characterization of retinoblastoma gene-mediated cell growth and tumor suppression in human cancer cells.
Proc Natl Acad Sci U S A
91
4165
4169
49. MaruoS
WuY
ItoT
KandaT
KieffED
2009
Epstein-Barr virus nuclear protein EBNA3C residues critical for maintaining lymphoblastoid cell growth.
Proc Natl Acad Sci U S A
106
4419
4424
50. JaresP
FernandezPL
CampoE
NadalA
BoschF
1994
PRAD-1/cyclin D1 gene amplification correlates with messenger RNA overexpression and tumor progression in human laryngeal carcinomas.
Cancer Res
54
4813
4817
51. BartkovaJ
LukasJ
MullerH
StraussM
GustersonB
1995
Abnormal patterns of D-type cyclin expression and G1 regulation in human head and neck cancer.
Cancer Res
55
949
956
52. BuckleyMF
SweeneyKJ
HamiltonJA
SiniRL
ManningDL
1993
Expression and amplification of cyclin genes in human breast cancer.
Oncogene
8
2127
2133
53. WilliamsME
SwerdlowSH
RosenbergCL
ArnoldA
1992
Characterization of chromosome 11 translocation breakpoints at the bcl-1 and PRAD1 loci in centrocytic lymphoma.
Cancer Res
52
5541s
5544s
54. MotokuraT
BloomT
KimHG
JuppnerH
RudermanJV
1991
A novel cyclin encoded by a bcl1-linked candidate oncogene.
Nature
350
512
515
55. LovecH
SewingA
LucibelloFC
MullerR
MoroyT
1994
Oncogenic activity of cyclin D1 revealed through cooperation with Ha-ras: link between cell cycle control and malignant transformation.
Oncogene
9
323
326
56. GaniatsasS
DowR
ThompsonA
SchulmanB
GermainD
2001
A splice variant of Skp2 is retained in the cytoplasm and fails to direct cyclin D1 ubiquitination in the uterine cancer cell line SK-UT.
Oncogene
20
3641
3650
57. YuZK
GervaisJL
ZhangH
1998
Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins.
Proc Natl Acad Sci U S A
95
11324
11329
58. AlaoJP
LamEW
AliS
BuluwelaL
BordognaW
2004
Histone deacetylase inhibitor trichostatin A represses estrogen receptor alpha-dependent transcription and promotes proteasomal degradation of cyclin D1 in human breast carcinoma cell lines.
Clin Cancer Res
10
8094
8104
59. PontanoLL
DiehlJA
2008
Speeding through cell cycle roadblocks: Nuclear cyclin D1-dependent kinase and neoplastic transformation.
Cell Div
3
12
60. SherrCJ
1996
Cancer cell cycles.
Science
274
1672
1677
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