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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

Microbiology > Microbial biochemistry > Protein > Modification
Authors: 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, 1202 views, 0 Q&A, How to cite
DOI: http://dx.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.

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.


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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