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Among the seven serines and one threonine in the carboxyl-terminus of HBV C protein, all but one (serine 183) appear in the context of RxxS/T consensus phosphoacceptor motifs and also overlap with other consensus motifs, such as S/TP, RS, SPRRR, RRRS/T, or RRxS/T, suggesting that various cellular kinases phosphorylate these residues. To determine whether threonine and/or serine (serines 157, 164, 170, 172, 178, and 180, and threonine 162, adw subtype) of HBV C protein are indeed phosphoacceptor residues in cells, Huh7 were transfected with a series of C-protein-expressing mutants, labeled with 32P-orthophosphate for 14 h, and then lysed. The 32Pi-labeled lysates were immunoprecipitated with anti-HBc antibody, and the 32Pi-labeled immunoprecipitated C proteins were detected by autoradiography.

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Detection of HBV C Protein Phosphorylation in the Cell
细胞中的HBV C蛋白磷酸化位点检测

微生物学 > 微生物生物化学 > 蛋白质 > 修饰
作者: Jaesung Jung
Jaesung JungAffiliation 1: Department of Microbiology, Ajou University School of Medicine, Suwon, Korea
Affiliation 2: Department of Biomedical Science, Graduate School of Ajou University, Suwon, Korea
Bio-protocol author page: a2449
 and Kyongmin Kim
Kyongmin KimAffiliation 1: Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea
Affiliation 2: Department of Biomedical Science, Graduate School of Ajou University, Suwon, Korea
Affiliation 3: Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea
For correspondence: kimkm@ajou.ac.kr
Bio-protocol author page: a2450
Vol 5, Iss 15, 8/5/2015, 2022 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1551

[Abstract] Among the seven serines and one threonine in the carboxyl-terminus of HBV C protein, all but one (serine 183) appear in the context of RxxS/T consensus phosphoacceptor motifs and also overlap with other consensus motifs, such as S/TP, RS, SPRRR, RRRS/T, or RRxS/T, suggesting that various cellular kinases phosphorylate these residues. To determine whether threonine and/or serine (serines 157, 164, 170, 172, 178, and 180, and threonine 162, adw subtype) of HBV C protein are indeed phosphoacceptor residues in cells, Huh7 were transfected with a series of C-protein-expressing mutants, labeled with 32P-orthophosphate for 14 h, and then lysed. The 32Pi-labeled lysates were immunoprecipitated with anti-HBc antibody, and the 32Pi-labeled immunoprecipitated C proteins were detected by autoradiography.

[Abstract]

Materials and Reagents

  1. Huh7 hepatoma cells (Japanese Collection of Research Bioresources Cell Bank, catalog number: JCRB0403 )
  2. Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Gibco®, catalog number: 12800-017 )
  3. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044 )
  4. Penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  5. Plasmid; HBV WT C protein (STSSSS) in plasmid HBV-P-def of pcDNA3 backbone, phosphoacceptor-site mutants (ATAAAA, AAAAAA, SSSSSS, and ASAAAA) in plasmid HBV-P-def in pcDNA3 backbone, pcDNA3-GFP
  6. Polyethylenimine (Polysciences, catalog number: 23966 )
  7. Opti-MEM (Life Technologies, Gibco®, catalog number: 31985-062 )
  8. OptiMEM (Life Technologies, Gibco®, catalog number: 31985-070 )
  9. Dialyzed FBS (Life Technologies, Gibco®, catalog number: 26400044 )
  10. 1 mCi orthophosphate [32Pi] (PerkinElmer Inc., catalog number: NEX053 )
  11. Polyclonal rabbit anti-HBc antibody (home-made, Jung et al., 2012)
  12. Protein A/G Plus agarose beads (Calbiochem®, catalog number: IP05 )
  13. Tris-HCl (pH 8.5) (Sigma-Aldrich, catalog number: T6066 )
  14. EDTA (Sigma-Aldrich, catalog number: E5134 )
  15. Nonidet P-40 (Sigma-Aldrich, catalog number: CA630 )
  16. NaF (Sigma-Aldrich, catalog number: 201154 )
  17. β-glycerophosphate (USB, catalog number: 155-56-2 )
  18. Sodium orthovanadate (Sigma-Aldrich, catalog number: s6508 )
  19. Protease inhibitors (Calbiochem®, catalog number: 535142 )
  20. Tris-HCl (pH 6.8) (Sigma-Aldrich, catalog number: T6066)
  21. SDS (Sigma-Aldrich, catalog number: L-3771 )
  22. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  23. Bromophenol blue (Sigma-Aldrich, catalog number: 114391 )
  24. Glycerol (USB, catalog number: 16374 )
  25. Lysis buffer (see Recipes)
  26. 2x sample buffer (see Recipes)

Equipment

  1. 10-cm dishes (Corning Incorporated, catalog number: 430167 )
  2. PVDF membranes (Bio-Rad Laboratories, AbD Serotec®, catalog number: 162-0177 )

Procedure

  1. Huh7 hepatoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under a humidified atmosphere at 37 °C in 5% CO2.
  2. Cells were passaged every three days (passage at 80~90% confluency) and 2 x 106 Huh7 cells were seeded in 10-cm dishes one day before the transfection.
  3. Next day, Huh7 cells were transfected using polyethylenimine (PEI).
    PEI transfection method:
    1. In a sterile tube, total 10 μg of plasmid DNA was mixed with 500 μl Opti-MEM.
    2. Add 30 μl of PEI solution (1 μg/1 μl) to DNA-Opti-MEM solution and then vortex immediately.
    3. Incubate 15 min at room temperature.
    4. Then add PEI/DNA-Opti-MEM mixture to cells.
    For transfections, 10 µg of plasmid encoding HBV STSSSS (WT) or phosphoacceptor-site mutant C proteins in a P-deficient mutant backbone were used. As a transfection control, 1 µg of green fluorescent protein (GFP)-expressing plasmid was included in the transfection mixture.
  4. Transfection experiments were repeated at least three times.
  5. Forty-eight hours after transfection, Huh7 cells were incubated at 37 °C in 5% CO2 incubator for 6 h in 10 ml DMEM containing 10% dialyzed FBS, then labeled at 37 °C in 5% CO2 for additional 14 h with 250 µCi orthophosphate [32Pi] per 107 cells, rinsed with ice-cold phosphate-buffered saline, and lysed with 1 ml of lysis buffer. (Isotope was added directly onto the cells containing DMEM-dFBS.)
  6. Lysates were transferred to 1.5 ml tube, incubated on ice for 10 min for further lysis, spin down at 13,000 rpm at 4 °C for 2 min, and supernatant was transferred to fresh 1.5 ml tube.
  7. The supernatant lysates were incubated for 2 h at 4 °C with 1 μl of polyclonal rabbit anti-HBc antibody on the rotator.
  8. Next, 15 μl of protein A/G Plus agarose beads was added, and the mixture was incubated at 4 °C for 1 h and centrifuged at 1,500 rpm for 10 sec. (Protein A/G Plus agarose bead preparation: Take 15 μl of bead/sample to fresh 1.5 ml tube, add 1 ml lysis buffer, vortex briefly, spin down at 1,500 rpm for 10 sec, and discard supernatant. Repeat above washing procedures 2 more times.)
  9. The pellets in 1.5 ml tube (containing beads bound to immune complexes of radiolabeled WT or mutant C proteins and anti-HBc antibodies) were washed twice with 1 ml lysis buffer, eluted by boiling (100 °C for 5 min) in 15 μl of 2x sample buffer, and separated by SDS-PAGE on 13.5% gels. Running time was 3 h at 80 voltage.
  10. Separated proteins were transferred to PVDF membranes (250 mA 1 h 30 min) and subjected to autoradiography.

Representative data



Compared to AAAAAA mutant of which the phosphoacceptor sites were all abolished to alanine, STSSSS (WT) and other mutants were all 32Pi-labeled.

Recipes

  1. Lysis buffer
    50 mM Tris-HCl (pH 8.5)
    2 mM EDTA
    0.5% Nonidet P-40
    50 mM NaF
    25 mM β-glycerophosphate
    2 mM sodium orthovanadate, and protease inhibitors
  2. 2x sample buffer
    100 mM Tris-HCl (pH 6.8)
    4% SDS
    200 mM β-mercaptoethanol
    0.2% bromophenol blue
    20% glycerol

Acknowledgments

This work was supported by National Research Foundation Grants funded by the Korean Government (NRF-2012-R1A2A2A01015370).

Reference

  1. Jung, J., Kim, H. Y., Kim, T., Shin, B. H., Park, G. S., Park, S., Chwae, Y. J., Shin, H. J. and Kim, K. (2012). C-terminal substitution of HBV core proteins with those from DHBV reveals that arginine-rich 167RRRSQSPRR175 domain is critical for HBV replication. PLoS One 7(7): e41087.
  2. Jung, J., Hwang, S. G., Chwae, Y. J., Park, S., Shin, H. J. and Kim, K. (2014). Phosphoacceptors threonine 162 and serines 170 and 178 within the carboxyl-terminal RRRS/T motif of the hepatitis B virus core protein make multiple contributions to hepatitis B virus replication. J Virol 88(16): 8754-8767.

材料和试剂

  1. Huh7肝癌细胞(Japanese Collection of Research Bioresources Cell Bank,目录号:JCRB0403)
  2. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:12800-017)
  3. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:16000-044)
  4. 青霉素/链霉素(Life Technologies,Gibco ,目录号:15140-122)
  5. 质粒; 在pcDNA3骨架中的质粒HBV-P-def中的pcDNA3-GFP的HBV WT C蛋白(STSSSS),在pcDNA3骨架中的质粒HBV-P-def中的pcDNA3主链的磷酸受体位点突变体(ATAAAA,AAAAAA,SSSSSS和ASAAAA)
  6. 聚乙烯亚胺(Polysciences,目录号:23966)
  7. Opti-MEM(Life Technologies,Gibco ,目录号:31985-062)
  8. OptiMEM(Life Technologies,Gibco ,目录号:31985-070)
  9. 透析的FBS(Life Technologies,Gibco ,目录号:26400044)
  10. 1 mCi正磷酸盐(PerkinElmer Inc.,目录号:NEX053)
  11. 多克隆兔抗HBc抗体(自制,Jung et al。,2012)
  12. 蛋白A/G Plus琼脂糖珠(Calbiochem ,目录号:IP05)
  13. Tris-HCl(pH8.5)(Sigma-Aldrich,目录号:T6066)
  14. EDTA(Sigma-Aldrich,目录号:E5134)
  15. Nonidet P-40(Sigma-Aldrich,目录号:CA630)
  16. NaF(Sigma-Aldrich,目录号:201154)
  17. β-磷酸甘油酯(USB,目录号:155-56-2)
  18. 原钒酸钠(Sigma-Aldrich,目录号:s6508)
  19. 蛋白酶抑制剂(Calbiochem ,目录号:535142)
  20. Tris-HCl(pH6.8)(Sigma-Aldrich,目录号:T6066)
  21. SDS(Sigma-Aldrich,目录号:L-3771)
  22. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  23. 溴酚蓝(Sigma-Aldrich,目录号:114391)
  24. 甘油(USB,目录号:16374)
  25. 裂解缓冲液(见配方)
  26. 2x样品缓冲液(见配方)

设备

  1. 10cm盘(Corning Incorporated,目录号:430167)
  2. PVDF膜(Bio-Rad Laboratories,AbD Serotec ,目录号:162-0177)

程序

  1. Huh7肝癌细胞在补充有10%胎牛血清和1%青霉素/链霉素的Dulbecco改良的Eagle's培养基(DMEM)中,在潮湿气氛下,在37℃,5%CO 2中生长。
  2. 细胞每三天传代(在80〜90%汇合时传代),并且在转染前一天将2×10 6个Huh7细胞接种在10-cm培养皿中。
  3. 第二天,使用聚乙烯亚胺(PEI)转染Huh7细胞。
    PEI转染方法:
    1. 在无菌试管中,将总共10μg的质粒DNA与500μlOpti-MEM混合。
    2. 加入30微升PEI溶液(1微克/1微升)到DNA-Opti-MEM溶液,然后立即涡旋。
    3. 在室温下孵育15分钟。
    4. 然后向细胞中加入PEI/DNA-Opti-MEM混合物。
    对于转染,使用10μg编码HBV缺失型突变体主链中的HBV STSSSS(WT)或磷酸受体位点突变体C蛋白的质粒。 作为转染对照,在转染混合物中包括1μg表达绿色荧光蛋白(GFP)的质粒。
  4. 将转染实验重复至少三次。
  5. 转染后48小时,Huh7细胞在37℃下在含有10%透析的FBS的10ml DMEM中的5%CO 2培养箱中孵育6小时,然后在37℃下在5% CO 2正常磷酸盐缓冲液再次培养14小时,每10 7个细胞用250μCi的正磷酸盐处理,用 冰冷的磷酸盐缓冲盐水中,并用1ml裂解缓冲液裂解。 (同位素直接加到含有DMEM-dFBS的细胞上)
  6. 将裂解物转移到1.5ml管中,在冰上孵育10分钟以进一步裂解,在4℃下以13,000rpm离心2分钟,将上清液转移到新鲜的1.5ml管中。
  7. 将上清液裂解物在旋转器上与1μl多克隆兔抗HBc抗体在4℃温育2小时。
  8. 接下来,加入15μl蛋​​白A/G Plus琼脂糖珠,将混合物在4℃下孵育1小时,并以1,500rpm离心10秒。 (蛋白A/G Plus琼脂糖珠制备:取15μl珠/样品至新鲜的1.5ml管中,加入1ml裂解缓冲液,短暂涡旋,以1,500rpm离心10秒,弃去上清液重复上述洗涤程序2更多次。)
  9. 1.5ml管中的沉淀(含有与放射性标记的WT或突变体C蛋白和抗HBc抗体的免疫复合物结合的珠子)用1ml裂解缓冲液洗涤两次,通过在15μl的乙腈中煮沸(100℃5分钟) 2x样品缓冲液,并通过在13.5%凝胶上的SDS-PAGE分离。在80电压下运行时间为3小时。
  10. 将分离的蛋白转移至PVDF膜(250mA 1小时30分钟),并进行放射自显影。

代表数据



与其中磷酸受体位点全部消除为丙氨酸的AAAAAA突变体相比,STSSSS(WT)和其他突变体都是32-标记的。

食谱

  1. 裂解缓冲液
    50mM Tris-HCl(pH8.5) 2mM EDTA 0.5%Nonidet P-40
    50mM NaF 25 mMβ-甘油磷酸盐 2mM原钒酸钠和蛋白酶抑制剂
  2. 2x样品缓冲液
    100 mM Tris-HCl(pH 6.8)
    4%SDS
    200mMβ-巯基乙醇 0.2%溴酚蓝
    20%甘油

致谢

这项工作得到了由韩国政府资助的国家研究基金会资助(NRF-2012-R1A2A2A01015370)的支持。

参考

  1. Jung,J.,Kim,H.Y.,Kim,T.,Shin,B.H.,Park,G.S.,Park,S.,Chwae,Y.J.,Shin,H.J.and Kim,K。 用DHBV的C端替代HBV核心蛋白揭示了富含精氨酸的167RRRSQSPRR175结构域是关键的 用于HBV复制。 PLoS One 7(7):e41087。
  2. Jung,J.,Hwang,S.G.,Chwae,Y.J.,Park,S.,Shin,H.J。和Kim,K。(2014)。 磷酸受体苏氨酸162和丝氨酸170和178在乙型肝炎的羧基末端RRRS/T基序内 病毒核心蛋白对乙型肝炎病毒复制有多重贡献。病毒核心蛋白对乙型肝炎病毒复制有多重贡献。 88(16):8754-8767。
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How to cite this protocol: Jung, J. and Kim, K. (2015). Detection of HBV C Protein Phosphorylation in the Cell. Bio-protocol 5(15): e1551. DOI: 10.21769/BioProtoc.1551; Full Text



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