目的 通過檢測異染色質(zhì)蛋白1α（HP1α）在DNA損傷后的磷酸化狀況，介紹一種用磷酸化標(biāo)簽(phos-tag)試劑檢測磷酸化蛋白質(zhì)的新方法。 方法 取雄雌C57小鼠交配后孕13.5 d胚胎，分離并原代培養(yǎng)小鼠胚胎成纖維細(xì)胞。對照組及實驗組(6個損傷時間點)各取2個100 mm培養(yǎng)皿的細(xì)胞進(jìn)行實驗，實驗組細(xì)胞用喜樹堿進(jìn)行DNA損傷；對照組用等量的二甲基亞砜處理。用摻入phos-tag的十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳分離蛋白并轉(zhuǎn)印，將膜用抗HP1α的抗體孵育，用偶聯(lián)辣根過氧化物酶的抗體做二抗，通過成像系統(tǒng)檢測蛋白。 結(jié)果 實驗組存在一條與HP1α有明顯不同遷移率的磷酸化HP1α條帶，與對照組相比DNA損傷后磷酸化HP1α含量一過性增多。 結(jié)論 HP1α被DNA損傷誘導(dǎo)為磷酸化狀態(tài)，提示其可能在DNA修復(fù)過程中扮演重要角色。 Phos-tag 蛋白質(zhì)印跡法可采用普通抗體檢測磷酸化的蛋白，是一種簡便易行的檢測未知磷酸化蛋白質(zhì)的新方法。
引用本文： 劉佳,周光前,尹獻(xiàn)輝,李雪芹,王優(yōu)雅,胡靜儀,王子梅. 磷酸化標(biāo)簽方法檢測異染色質(zhì)蛋白1αDNA損傷后蛋白磷酸化狀態(tài). 華西醫(yī)學(xué), 2012, 27(6): 828-832. doi: 復(fù)制
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| 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. |
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| 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. |
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- 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.