• 1四川大學華西醫(yī)院生物治療國家重點實驗室（成都，610041）;2深圳大學醫(yī)學院生物化學與分子生物學教研室;

目的 　通過檢測異染色質蛋白1α（HP1α）在DNA損傷后的磷酸化狀況，介紹一種用磷酸化標簽(phos-tag)試劑檢測磷酸化蛋白質的新方法。 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)皿的細胞進行實驗，實驗組細胞用喜樹堿進行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。 結論 　HP1α被DNA損傷誘導為磷酸化狀態(tài)，提示其可能在DNA修復過程中扮演重要角色。 Phos-tag 蛋白質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的新方法。

引用本文： 劉佳,周光前,尹獻輝,李雪芹,王優(yōu)雅,胡靜儀,王子梅. 磷酸化標簽方法檢測異染色質蛋白1αDNA損傷后蛋白磷酸化狀態(tài). 華西醫(yī)學, 2012, 27(6): 828-832. doi: 復制

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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.
  1. 1. 　Hunter T. Signaling-2000 and beyond[J]. Cell, 2000, 100(1): 113-127.
  2. 2. 　Pandey A, Mann M. Proteomics to study genes and genomes[J]. Nature, 2000, 405(6788): 837-846.
  3. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 17. 　Downey M, Durocher D. Chromatin and DNA repair: the benefits of relaxation[J]. Nat Cell Biol, 2006, 8(1): 9-10.
  18. 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. 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.