摘要
在整个周期进程中,细胞顺利完成G_2/M期的转换是实现细胞增殖的一个关键步骤。由于细胞自身存在G_2期DNA损伤检验点的监控机制,所以可以保证G_2/M期转换的顺利实现。由于Polo like激酶(Plk1)和p53分别是通过正、负调控机制在G_2/M转换过程发挥重骂的作用,并且有研究表明Plk1可以参与调节p53的活性。因此,本文致力研究Plk1是否可以通过调节p53蛋白参与G_2/M期转换过程的调控。主要结果如下:
1.将同步化在G_2期的细胞释放培养,Plk1的激酶活性上升,说明细胞由G_2期顺利地向M期过渡。采用低剂量UV照射的方法处理同步化在G_2期的细胞,使Plk1的激酶活性受到抑制,并且伴随着蛋白表达水平的降低,而p53的转录活性升高、蛋白大量积聚。
2.通过细胞免疫共沉淀实验证实,在G2/M期Plk1与p53发生物理性的结合。酵母双杂交实验进一步确证Plk1与p53的DNA结合区域发生直接结合作用。表明Plk1可能是通过竞争性结合p53的DNA结合区域来抑制p53转录活性。在细胞内过表达Plk1也证明p53的转录活性受到抑制。
3.细胞内过表达Plk1可以抑制p53蛋白的磷酸化;共同过表达Plk1和Mdm2可以加剧Mdm2介导p53的泛素化降解;过表达Plk1还可以促使p53蛋白被转运出细胞核。表明Plk1可以通过抑制p53的磷酸化、促进p53蛋白降解以及抑制p53的入核作用来影响p53的蛋白稳定性和转录活性。
4.Plk1和hNRAGE分别作用于p53的不同区域,Plk1抑制p53的转录活性,而hNRAGE上调p53的转录活性,当两者共同作用后会相互抵消对p53的作用。hNRAGE对Plk1的激酶活性没有影响。表明Plk1和hNRAGE可能作为两个独立的信号分子参与调节p53。
During the cell cycle, successful accomplishment of G_2/M transition is a pivotal process to carry out proliferation. Cell itself has the surveillant mechanism at G_2 damage checkpoint, which ensures the successful accomplishment of G_2/M transition. Polo like kinase (Plk1) regulates the G_2/M transition by the positive mechanism while p53 by the negative one. Researches showed that Plk1 paticipated in regulating the activation of p53. So we wanted to find out whether Plkl regulates the G_2/M transition via p53 pathway in this article. Main results are listed below:1. When the synchronized cells released from the G_2 phase, Plkl kinase activity increased. It meant that the cell cycle could transform from G_2 phase to M phase. When those synchronized cells were treated with low dose UV, kinase activity of endogenous Plk1 significantly was down-regulated and its protein level decreased while transactivation of p53 remarkably was up-regulated with its protein level accumulation.2. We comfirmed by immunoprecipitation that Plk1 could physically interact with p53 at the G_2/M transitional phase. The Gal-4 two-hybrid system further indicated that Plk1 directly interacted with the sequence-specific DNA-binding domain of p53. That is to say Plkl might restrain the transactivation of p53 by competitivly binding with the sequence-specific DNA-binding domain of p53. It could be testified by the experiment that over-expression of Plk1 in cultured cells could inhibit the transactivation of p53.3. Over-expression of Plk1 in cultured cells could inhibit the phosphorylation of p53 protein. Over-expression of Plk1 accompanied with Mdm2 could accelerate Mdm2 processing degradation of p53. At the same time, over-expression of Plk1 could inhibit processing nuclear import of p53. These results indicated that Plk1 also leaded to the reduction of stability and transactivation of p53 by inhibiting its phosphorylation, accerating its degradation and preventing its nuclear import.
4. Plkl and hNRAGE interacted with the different domains of p53 respectively. Plkl negatively mediated the transactivational activity of p53, while the hNRAGE positivly regulated p53 transactivational activity. When they acted together, their influence to p53 counteracted with each other. hNRAGE did not effect the kinase activity of Plkl. So Plkl and hNRAGE might be the two relatively independent cell signals that regulate the activation of p5 3.As a result, in the G2 damage checkpoint pathway, Plkl regulates the stability and transactivation of p53 by binding to the sequence-specific DNA-binding domain of p53, inhibiting its phosphorylation, promoting its degradation and preventing its nuclear importing. Plkl can paticipate in regulating the G2/M transition via p53 pathway through negatively regulating its transactivational activity.
引文
1. Glover DM, Hagan IM, Tavares AA. Polo-like kinases: a team that plays throughout mitosis. Genes Dev. 1998 Dec 15; 12(24): 3777-87.
2. Jang Y J, Lin CY, Ma S, Erikson RL. Functional studies on the role of the C-terminal domain of mammalian polo-like kinase. Proe Natl Acad Sci USA. 2002 Feb 19; 99(4): 1984-9
3. Hamanaka R, Smith MR, O'Connor PM, Maloid S, Mihalic K, Spivak JL, Longo DL, Ferris DK. Polo-like kinase is a cell cycle-regulated kinase activated during mitosis J Biol Chem. 1995 Sep 8; 270(36): 21086-91
4. Lee KS, Grenfell TZ, Yarm FR, Erikson RL. Mutation of the polo-box disrupts localization and mitotic functions of the mammalian polo kinase Plk. Proc. Nat. Acad. Sci. USA 1998, 95(16): 9301-9306
5. Bahassi el M, Conn CW, Myer DL, Hennigan RF, McGowan CH, Sanchez Y, Stambrook PJ. Mammalian Polo-like kinase 3 (Plk3) is a multifunctional protein involved in stress response pathways. Oncogene. 2002 Sep 26; 21(43): 6633-40.
6. Roshak AK, Capper EA, Imburgia C, Fornwald J, Scott G, Marshall LA. The human polo-like kinase, PLK, regulates cdc2/CyclinB through phosphorylation and activation of the cdc25C phosphatase. Cell Signal. 2000 Jun; 12(6): 405-11.
7. Lane HA, Nigg EA. Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes. J Cell Biol. 1996 Dec; 135(6 Pt 2): 1701-13.
8. Liu X, Erikson RL. Activation of Cdc2/cyclin B and inhibition of centrosome amplification in cells depleted of Plk1 by siRNA. Proc Natl Acad Sci USA. 2002 Jun 25; 99(13): 8672-6.
9. Sudakin V, Ganoth D, Dahan A, Heller H, Hershko J, Luca FC, Ruderman JV, Hershko A. The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. Mol Biol Cell. 1995 Feb; 6(2): 185-97.
10. Golan A, Yudkovsky Y, Hershko A. The Cyclin-Ubiquitin Ligase Activity of Cyclosome/APC Is Jointly Activated by Protein Kinases Cdk1-CyclinB and Plk. J Biol Chem 2002 May 3; 277(18): 15552-7.
11. Kotani S, Tugendreich S, Fujii M, Jorgensen PM, Watanabe N, Hoog C, Hieter P, Todokoro K. PKA and MPF-activated polo-like kinase regulate anaphaseopromoting complex activity and mitosis progression. Mol Cell. 1998 Feb; 1(3): 371-80
12. Zachariae W, Nasmyth K. Whose end is destruction: cell division and the anaphase-promoting complex. Genes Dev. 1999 Aug 15; 13 (16): 2039-58.
13. Neef R, Preisinger C, Sutcliffe J, Kopajtich R, Nigg EA, Mayer TU, Barr FA. Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis. J Cell Biol. 2003 Sep 1; 162(5): 863-75.
14. Mulvihill DP and Hyams JS. Cytokinetic actomyosin ring formation and septation in fission yeast are dependent on the full recruitment of the polo-like kinase Plol to the spindle pole body and a functional spindle assembly checkpoint. J Cell Sci. 2002 Sep 15; 115(18): 3575-86.
15. Clute P, Pines J. Temporal and spatial control of CyclinB1 destruction in metaphase. Nat Cell Biol. 1999 Jun; 1(2): 82-7
16. Hagting A, Den Elzen N, Vodermaier HC, Waizenegger IC, Peters JM, Pines J. Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1. J Cell Biol. 2002 Jun 24; 157(7): 1125-37.
17. Lindon C, Pines J. Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells. J Cell Biol. 2004 Jan 19; 164(2): 233-241
18. Carmena M, Riparbelli MG, Minestrini G, Tavares AM, Adams R, Callaini G, Glover DM. Drosophila polo kinase is required for cytokinesis. J Cell Biol. 1998 Nov 2; 143(3): 659-71.
19. Heitz MJ, Petersen J, Valovin S, Hagan IM. MTOC formation during mitotic exit in fission yeast. J Cell Sci. 2001 Dec; 114(Pt 24): 4521-32
20. Song S, Lee KS. A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis. J Cell Biol. 2001 Feb 5; 152(3): 451-69.
21. Seong YS, Kamijo K, Lee JS, Fernandez E, Kuriyama R, Miki T, Lee KS. A spindle checkpoint arrest and a cytokinesis failure by the dominant-negative polo-box domain of Plk1 in U-2OS cells. J Biol Chem. 2002 Aug 30; 277(35): 32282-93.
22. Arnaud L, Pines J, Nigg EA. GFP tagging reveals human Polo-like kinase 1 at the kinetochore/centromere region of mitotic chromosomes. Chromosoma. 1998 Dec; 107(6-7): 424-9
23. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997 Feb 7; 88(3):323-31.
24. Boyd SD, Tsai KY, Jacks T. An intact HDM2 RING-finger domain is required for nuclear exclusion of p53. Nat Cell Biol. 2000 Sep; 2(9): 563-8.
25. Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 1997 Dec 22; 420(1): 25-7.
26. Momand J, Zambetti GP, Olson DC, George D, Levine AJ. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell. 1992 Jun 26; 69(7): 1237-45.
27. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature. 2000 Nov 16; 408(6810): 307-10.
28. Sharpless NE and DePinho RA. p53: good cop/bad cop. Cell. 2002 Jul 12; 110(1): 9-12.
29. Lohrum M, Scheidtmann KH. Differential effects of phosphorylation of rat p53 on transactivation of promoters derived from different p53 responsive genes. Oneogene. 1996 Dec 19; 13(12): 2527-39.
30. Chehab NH, Malikzay A, Stavridi ES, Halazonetis TD. Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage. Proc Natl Acad Sci USA. 1999 Nov 23; 96(24): 13777-82
31. Keller DM, Lu H. p53 serine 392 phosphorylation increases after UV through induction of the assembly of the CK2.hSPT16.SSRP1 complex. J Biol Chem. 2002 Dec 20; 277(51): 50206-13.
32. Shieh SY, Ikeda M, Taya Y, Prives C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell. 1997 Oct 31; 91(3): 325-34.
33. el-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B. Definition of a consensus binding site for p53. Nat. Genet. (1992)1: 45-49
34. Tang D, Wu D, Hirao A, Lahti JM, Liu L, Mazza B, Kidd V J, Mak TW, Ingrain AJ. ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53. J Biol Chem. 2002 Apr 12; 277(15): 12710-7.
35. Smith GC, Cary RB, Lakin ND, Hann BC, Teo SH, Chen DJ, Jackson SP. Purification and DNA binding properties of the ataxiatelangiectasia gene product ATM. Proc Natl Acad Sci USA. 1999; 96: 11134-11139.
36. Suzuki K, Kodama S, Watanabe M. Recruitment of ATM protein to double strand DNA irradiated with ionizing radiation. J Biol Chem. 1999; 274: 25571-25575.
37. Guo Z, Kumagai A, Wang SX, Dunphy WG. Eequirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes Dev. 2000 Nov 1; 14(21): 2745-56
38. Helt CE, Cliby WA, Keng PC, Bambara RA, O'Reilly MA. Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related protein exhibit selective target specificities in response to different forms of DNA damage. J Biol Chem, 2005 Jan 14; 280(2): 1186-92.
39. Zhao H, Piwnica WH. ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Boil. 2001; 21: 4129-4139.
40. Alan JY, Li X, Davis HL. Canman CE. Phosphorylation of threonine 68 promotes oligomerization and autophosphorylation of the Chk2. J Biol Chem. 2002 May 31; 277(22): 19389-95
41. Matsuoka S, Huang M, Elledge SJ. Lindage of ATM to cell cycle regulation by the Chk2 protein kinase. Science. 1998; 282: 1893-1897.
42. Kang D, Chen J, Wong J, Fang G. The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition. J Cell Biol. 2002 Jan 21; 156(2): 249-59.
43. Scolnick DM, Halazonetis TD. Chfr defines a mitotic stress checkpoint that delays entry into metaphase. Nature. 2000 Jul 27; 406(6794): 430-5.
44. Smits VA, Klompmaker R, Arnaud L, Rijksen G, Nigg EA, Medema RH. Polo-like kinase-1 is a target of the DNA damage checkpoint. Nat Cell Biol. 2000 Sep; 2(9): 672-6.
45. Liu X, Erikson RL. Polo-like kinase (Plk)1 depletion induces apoptosis in cancer cells. Proc Natl Acad Sci. 2003 May 13; 100(10): 5789-94.
46. van Vugt MA, Smits VA, Klompmaker R, Medema RH. Inhibition of Polo-like Kinase-1 by DNA Damage Occurs in an ATM-or ATR-dependent Fashion. J Biol Chem. 2001 Nov 9; 276(45): 41656-60.
47. Ree AH, Bratland A, Solberg Landsverk K, Fodstad O. Ionizing radiation inhibits the PLK cell cycle gene in a G_2 checkpoint-dependent manner. Anticancer Res. 2004 Mar-Apr; 24(2B): 555-62
48. Winters ZE. P53 pathways involving G2 checkpoint regulators and the role of their subcellular localisation. J R Coll Surg Edinb. 2002 Aug; 47(4): 591-8.
49. Lesser MP, Kruse VA, Barry TM. Exposure to ultraviolet radiation causes apoptosis in developing sea urchin embryos. J Exp Biol. 2003 Nov; 206(22): 4097-103.
50. Maeda T, Chong MT, Espino RA, Chua PP, Cao JQ, Chomey EG, Luong L, Tron VA. Role of p21(Waf-1) in regulating the G_1 and G_2/M checkpoints in ultraviolet-irradiated keratinocytes. J Invest Dermatol. 2002 Aug; 119(2): 513-21.
51. Sanchez Y, Elledge SJ. Stopped for repairs. Bioessays. 1995 Jun; 17(6): 545-8.
52. Maity A, McKenna WG, Muschel RJ. The molecular basis for cell cycle delays following ionizing radiation: a review. Radiother Oncol. 1994 Apr; 31(1): 1-13.
53. Maya R, Balass M, Kim ST, Shkedy D, Leal JF, Shifman O, Moas M, Buschmann T, Ronai Z, Shiloh Y, Kastan MB, Katzir E, Oren M. ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev. 2001 May 1; 15(9): 1067-77.
54. Sanchez Y, Wong C, Thoma RS, Richman R, Wu Z, Piwnica-Worms H, Elledge SJ. Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science. 1997 Sep 5; 277(5331): 1497-501.
55. O'Connell MJ, Walworth NC, Carr AM. The G2-phase DNA-damage checkpoint. Trends Cell Biol. 2000 Jul; 10(7): 296-303.
56. Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ. Chk1 is an essential kinase that is regulated by Atr and required for the G_2/M DNA damage checkpoint. Genes Dev. 2000 Jun 15; 14(12): 1448-59.
57. Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20; 340(6230): 245-6.
58. Fields S, Sternglanz R. The two-hybrid system: An assay for protein-protein interactions. Trends Genet. 1994 Aug; 10(8): 286-92.
59. Brachmann RK and Boeke JD. Tag games in yeast: the two-hybrid system and beyond. Curr Opin Biotechnol. 1997 Oct; 8(5): 561-8.
60. Ando K, Ozaki T, Yamamoto H, Furuya K, Hosoda M, Hayashi S, Fukuzawa M, Nakagawara A. Polo-like Kinase 1 (Plk1) Inhibits p53 Function by Physical Interaction and Phosphorylation. J Biol Chem. 2004 Jun 11; 279(24): 25549-61.
61. Salehi AH, Roux PP, Kubu CJ, Zeindler C, Bhakar A, Tannis LL, Verdi JM, Barker PA. NRAGE, a novel MAGE protein, interacts with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis. Neuron. 2000 Aug; 27(2): 279-88.
62. Aloyz RS, Bamji SX, Pozniak CD, Toma JG, Atwal J, Kaplan DR, Miller FD. p53 is essential for developmental neuron death as regulated by the TrkA and p75 neurotrophin receptors. J Cell Biol. 1998 Dec 14; 143(6): 1691-703.
63. Bhakar AL, Howell JL, Paul CE, Salehi AH, Becker EB, Said F, Bonni A, Barker PA. Apoptosis induced by p75NTR overexpression requires Jun kinase-dependent phosphorylation of Bad. J Neurosci. 2003 Dec 10; 23(36): 11373-81.
64. Jordan BW, Dinev D, LeMellay V, Troppmair J, Gotz R, Wixler L, Sendtner M, Ludwig S, Rapp UR. Neurotrophin receptor-interacting mage homologue is an inducible inhibitor of apoptosis protein-interacting protein that augments cell death. J Biol Chem. 2001 Oct 26; 276(43): 39985-9.
65. Wen CJ, Xue B, Qin WX, Yu M, Zhang MY, Zhao DH, Gao X, Gu JR, Li CJ. Hnrage, a human neurotrophin receptor interacting MAGE homologue, regulates p53 transcriptional activity and inhibits cell proliferation. FEBS Lett. 2004 Apr 23; 564(1-2): 171-6.