Phos-tag Based Analysis of Heterochromatin Protein-1α Phosphorylation after DNA Damage_West China Medical Journal

Authors：

LIU Jia ¹ , ZHOU Guangqian ^1,2 , YIN Xianhui ² , LI Xueqin ² , WANG Youya ² , HU Jingyi ² ,  WANG Zimei ²

1. State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China; 2. School of Medicine, Shenzhen University, Shenzhen, Guangdong 518060, P. R. China;

Corresponding author：

WANG Zimei, Email: liujia-0628@163.com

Keywords：

蛋白质磷酸化; 磷酸化标签; 异染色质蛋白1α; DNA损伤

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目的　通过检测异染色质蛋白1α（HP1α）在DNA损伤后的磷酸化状况，介绍一种用磷酸化标签(phos-tag)试剂检测磷酸化蛋白质的新方法。方法　取雄雌C57小鼠交配后孕13.5 d胚胎，分离并原代培养小鼠胚胎成纤维细胞。对照组及实验组(6个损伤时间点)各取2个100 mm培养皿的细胞进行实验，实验组细胞用喜树碱进行DNA损伤；对照组用等量的二甲基亚砜处理。用掺入phos-tag的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离蛋白并转印，将膜用抗HP1α的抗体孵育，用偶联辣根过氧化物酶的抗体做二抗，通过成像系统检测蛋白。结果　实验组存在一条与HP1α有明显不同迁移率的磷酸化HP1α条带，与对照组相比DNA损伤后磷酸化HP1α含量一过性增多。结论　HP1α被DNA损伤诱导为磷酸化状态，提示其可能在DNA修复过程中扮演重要角色。 Phos-tag 蛋白质印迹法可采用普通抗体检测磷酸化的蛋白，是一种简便易行的检测未知磷酸化蛋白质的新方法。

Citation： LIU Jia,ZHOU Guangqian,YIN Xianhui,LI Xueqin,WANG Youya,HU Jingyi,WANG Zimei. Phos-tag Based Analysis of Heterochromatin Protein-1α Phosphorylation after DNA Damage. West China Medical Journal, 2012, 27(6): 828-832. doi: Copy

1.	Hunter T. Signaling-2000 and beyond[J]. Cell, 2000, 100(1): 113-127.
2.	Pandey A, Mann M. Proteomics to study genes and genomes[J]. Nature, 2000, 405(6788): 837-846.
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8.	Kinoshita E, Kinoshita-Kikuta E, Takiyama K, et al. Phosphate-binding tag, a new tool to visualize phosphorylated proteins[J]. Mol Cell Proteomics, 2006, 5(4): 749-757.
9.	Ayoub N, Jeyasekharan AD, Bernal JA, et al. HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response[J]. Nature, 2008, 453(7195): 682-686.
10.	Goodarzi AA, Noon AT, Deckbar D, et al. ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin[J]. Mol Cell, 2008, 31(2): 167-177.
11.	Liu B, Wang J, Chan K M, et al. Genomic instability in laminopathy-based premature aging[J]. Nat Med, 2005, 11(7): 780-785.
12.	Wu CC, Maccoss MJ. Shotgun proteomics: tools for the analysis of complex biological systems[J]. Curr Opin Mol Ther, 2002, 4(3): 242-250.
13.	Krishnan V, Chow MZ, Wang Z, et al. Histone H4 lysine 16 hypoacetylation is associated with defective DNA repair and premature senescence in Zmpste24-deficient mice[J]. Proc Natl Acad Sci USA, 2011, 108(30): 12325-12330.
14.	Baldeyron C, Soria G, Roche D, et al. HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair[J]. J Cell Biol, 2011, 193(1): 81-95.
15.	Cann KL, Dellaire G. Heterochromatin and the DNA damage response: the need to relax[J]. Biochem Cell Biol, 2011, 89(1): 45-60.
16.	Luijsterburg MS, Dinant C, Lans H, et al. Heterochromatin protein 1 is recruited to various types of DNA damage[J]. J Cell Biol, 2009, 185(4): 577-586.
17.	Downey M, Durocher D. Chromatin and DNA repair: the benefits of relaxation[J]. Nat Cell Biol, 2006, 8(1): 9-10.
18.	Hiragami-Hamada K, Shinmyozu K, Hamada D, et al. N-terminal phosphorylation of HP1alpha promotes its chromatin binding[J]. Mol Cell Biol, 2011, 31(6): 1186-1200.
19.	Minc E, Allory Y, Worman HJ, et al. Localization and phosphorylation of HP1 proteins during the cell cycle in mammalian cells[J]. Chromosoma, 1999, 108(4): 220-234.

1. 　Hunter T. Signaling-2000 and beyond[J]. Cell, 2000, 100(1): 113-127.
2. 　Pandey A, Mann M. Proteomics to study genes and genomes[J]. Nature, 2000, 405(6788): 837-846.
3. 　Cohen P. Protein kinases-the major drug targets of the twenty-first century?[J]. Nat Rev Drug Discov, 2002, 1(4): 309-315.
4. 　Takeda H, Kawasaki A, Takahashi M, et al. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule[J]. Rapid Commun Mass Spectrom, 2003, 17(18): 2075-2081.
5. 　Kinoshita E, Kinoshita-Kikuta E, Koike T. Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE[J]. Nat Protoc, 2009, 4(10): 1513-1521.
6. 　Aguilar HN, Tracey CN, Tsang SC, et al. Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes[J]. PLoS One, 2011, 6(6): 20903.
7. 　Kinoshita E, Kinoshita-Kikuta E, Koike T. Phos-tag SDS-PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral pH conditions[J]. Proteomics, 2012, 12(2): 192-202.
8. 　Kinoshita E, Kinoshita-Kikuta E, Takiyama K, et al. Phosphate-binding tag, a new tool to visualize phosphorylated proteins[J]. Mol Cell Proteomics, 2006, 5(4): 749-757.
9. 　Ayoub N, Jeyasekharan AD, Bernal JA, et al. HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response[J]. Nature, 2008, 453(7195): 682-686.
10. 　Goodarzi AA, Noon AT, Deckbar D, et al. ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin[J]. Mol Cell, 2008, 31(2): 167-177.
11. 　Liu B, Wang J, Chan K M, et al. Genomic instability in laminopathy-based premature aging[J]. Nat Med, 2005, 11(7): 780-785.
12. 　Wu CC, Maccoss MJ. Shotgun proteomics: tools for the analysis of complex biological systems[J]. Curr Opin Mol Ther, 2002, 4(3): 242-250.
13. 　Krishnan V, Chow MZ, Wang Z, et al. Histone H4 lysine 16 hypoacetylation is associated with defective DNA repair and premature senescence in Zmpste24-deficient mice[J]. Proc Natl Acad Sci USA, 2011, 108(30): 12325-12330.
14. 　Baldeyron C, Soria G, Roche D, et al. HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair[J]. J Cell Biol, 2011, 193(1): 81-95.
15. 　Cann KL, Dellaire G. Heterochromatin and the DNA damage response: the need to relax[J]. Biochem Cell Biol, 2011, 89(1): 45-60.
16. 　Luijsterburg MS, Dinant C, Lans H, et al. Heterochromatin protein 1 is recruited to various types of DNA damage[J]. J Cell Biol, 2009, 185(4): 577-586.
17. 　Downey M, Durocher D. Chromatin and DNA repair: the benefits of relaxation[J]. Nat Cell Biol, 2006, 8(1): 9-10.
18. 　Hiragami-Hamada K, Shinmyozu K, Hamada D, et al. N-terminal phosphorylation of HP1alpha promotes its chromatin binding[J]. Mol Cell Biol, 2011, 31(6): 1186-1200.
19. 　Minc E, Allory Y, Worman HJ, et al. Localization and phosphorylation of HP1 proteins during the cell cycle in mammalian cells[J]. Chromosoma, 1999, 108(4): 220-234.

West China Medical Journal

Phos-tag Based Analysis of Heterochromatin Protein-1α Phosphorylation after DNA Damage

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