Skip to main content

The NTPase/helicase domain of hepatitis C virus nonstructural protein 3 inhibits protein kinase C independently of its NTPase activity

Abstract

Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3, to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro. PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all of the PKC isotypes were inhibited with IC50-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCα was inhibited with a micromolar IC50 in this assay, which compares well with our result for NS3h-D1316A (IC50 = 0.7 μM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.

Abbreviations

ATP:

adenosine triphosphate

CREB:

cAMP response elementbinding protein

ER:

endoplasmic reticulum

HBV:

hepatitis B virus

HCC:

hepatocellular carcinoma

IFN-α:

alpha-interferon

Km:

Michaelis constant

Pi:

inorganic phosphate

PKA:

cAMP-dependent protein kinase

PKC:

protein kinase C

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

STAT:

signal transducer and activator of transcription

References

  1. El-Serag, H.B. and Rudolph, K.L. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132 (2007) 2557–2576.

    Article  CAS  PubMed  Google Scholar 

  2. Borowski, P., Heiland, M., Oehlmann, K., Becker, B., Kornetzky, L., Feucht, H. and Laufs, R. Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase. Eur. J. Biochem. 237 (1996) 611–618.

    Article  CAS  PubMed  Google Scholar 

  3. Borowski, P., Resch, K., Schmitz, H. and Heiland, M. A synthetic peptide derived from the non-structural protein 3 of hepatitis C virus serves as a specific substrate for PKC. Biol. Chem. 381 (2000) 19–27.

    Article  CAS  PubMed  Google Scholar 

  4. Borowski, P., Kuhl, R., Laufs, R., Schulze zur Wiesch, J. and Heiland, M. Identification and characterization of a histone binding site of the nonstructural protein 3 of hepatitis C virus. J. Clin. Virol. 13 (1999) 61–69.

    Article  CAS  PubMed  Google Scholar 

  5. Borowski, P., Schulze zur Wiesch, J., Resch, K., Feucht, H., Laufs, R. and Schmitz, H. Protein kinase C recognizes the protein kinase A-binding motif of nonstructural protein 3 of hepatitis C virus. J. Biol. Chem. 274 (1999) 30722–30728.

    Article  CAS  PubMed  Google Scholar 

  6. Hartjen, P., Medom, B.K., Reinholz, M., Borowski, P. and Baier, A. Regulation of the biochemical function of motif VI of HCV NTPase/helicase by the conserved Phe-loop. Biochimie 91 (2009) 252–260.

    Article  CAS  PubMed  Google Scholar 

  7. Borowski, P., Oehlmann, K., Heiland, M. and Laufs, R. Nonstructural protein 3 of hepatitis C virus blocks the distribution of the free catalytic subunit of cyclic AMP-dependent protein kinase. J. Virol. 71 (1997) 2838–2843.

    CAS  PubMed  Google Scholar 

  8. Kemp, B.E. and Pearson, R.B. Design and use of peptide substrates for protein kinases. Methods Enzymol. 200 (1991) 121–134.

    Article  CAS  PubMed  Google Scholar 

  9. House, C. and Kemp, B.E. Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science 238 (1987) 1726–1728.

    Article  CAS  PubMed  Google Scholar 

  10. Aoubala, M., Holt, J., Clegg, R.A., Rowlands, D.J. and Harris, M. The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis. J. Gen. Virol. 82 (2001) 1637–1646.

    CAS  PubMed  Google Scholar 

  11. Griner, E.M. and Kazanietz, M.G. Protein kinase C and other diacylglycerol effectors in cancer. Nat. Rev. Cancer 7 (2007) 281–294.

    Article  CAS  PubMed  Google Scholar 

  12. Kim, D.W., Gwack, Y., Han, J.H. and Choe, J. C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem. Biophys. Res. Commun. 215 (1995) 160–166.

    Article  CAS  PubMed  Google Scholar 

  13. Wardell, A.D., Errington, W., Ciaramella, G., Merson, J. and McGarvey, M.J. Characterization and mutational analysis of the helicase and NTPase activities of hepatitis C virus full-length NS3 protein. J. Gen. Virol. 80 (Pt 3) (1999) 701–709.

    Google Scholar 

  14. Tai, C.L., Chi, W.K., Chen, D.S. and Hwang, L.H. The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). J. Virol. 70 (1996) 8477–8484.

    CAS  PubMed  Google Scholar 

  15. Persistence of Vision Pty. Ltd. (2004): Persistence of Vision Raytracer (Version 3.6) [Computer software]. Retrieved from http://www.povray.org/download/.

    Google Scholar 

  16. Yao, N., Hesson, T., Cable, M., Hong, Z., Kwong, A.D., Le, H.V. and Weber, P.C. Structure of the hepatitis C virus RNA helicase domain. Nat. Struct. Biol. 4 (1997) 463–467.

    Article  CAS  PubMed  Google Scholar 

  17. Kholodenko, B., Zilinskiene, V., Borutaite, V., Ivanoviene, L., Toleikis, A. and Praskevicius, A. The role of adenine nucleotide translocators in regulation of oxidative phosphorylation in heart mitochondria. FEBS Lett. 223 (1987) 247–250.

    Article  CAS  PubMed  Google Scholar 

  18. Dixon, M. The determination of enzyme inhibitor constants. Biochem. J. 55 (1953) 170–171.

    CAS  PubMed  Google Scholar 

  19. Cornish-Bowden, A. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J. 137 (1974) 143–144.

    CAS  PubMed  Google Scholar 

  20. Borowski, P., Heiland, M., Feucht, H. and Laufs, R. Characterisation of nonstructural protein 3 of hepatitis C virus as modulator of protein phosphorylation mediated by PKA and PKC: evidences for action on the level of substrate and enzyme. Arch. Virol. 144 (1999) 687–701.

    Article  CAS  PubMed  Google Scholar 

  21. Fimia, G.M., Evangelisti, C., Alonzi, T., Romani, M., Fratini, F., Paonessa, G., Ippolito, G., Tripodi, M. and Piacentini, M. Conventional protein kinase C inhibition prevents alpha interferon-mediated hepatitis C virus replicon clearance by impairing STAT activation. J. Virol. 78 (2004) 12809–12816.

    Article  CAS  PubMed  Google Scholar 

  22. Chen, J., Wu, M., Zhang, X., Zhang, W., Zhang, Z., Chen, L., He, J., Zheng, Y., Chen, C., Wang, F., Xu, Y., Lu, M. and Yuan, Z. Hepatitis B virus polymerase impairs interferon-alpha-induced STA T activation through inhibition of importin-alpha5 and protein kinase C-delta. Hepatology 57 (2013) 470–482.

    Article  CAS  PubMed  Google Scholar 

  23. Smith, B.L., Krushelnycky, B.W., Mochly-Rosen, D. and Berg, P. The HIV nef protein associates with protein kinase C theta. J. Biol. Chem. 271 (1996) 16753–16757.

    Article  CAS  PubMed  Google Scholar 

  24. Tardif, M., Savard, M., Flamand, L. and Gosselin, J. Impaired protein kinase C activation/translocation in Epstein-Barr virus-infected monocytes. J. Biol. Chem. 277 (2002) 24148–24154.

    Article  CAS  PubMed  Google Scholar 

  25. Volmer, R., Monnet, C. and Gonzalez-Dunia, D. Borna disease virus blocks potentiation of presynaptic activity through inhibition of protein kinase C signaling. PLoS Pathog. 2 (2006) e19.

    Article  PubMed  Google Scholar 

  26. Oster, H. and Leitges, M. Protein kinase C alpha but not PKCzeta suppresses intestinal tumor formation in ApcMin/+ mice. Cancer Res. 66 (2006) 6955–6963.

    Article  CAS  PubMed  Google Scholar 

  27. Wolk, B., Sansonno, D., Krausslich, H.G., Dammacco, F., Rice, C.M., Blum, H.E. and Moradpour, D. Subcellular localization, stability, and transcleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines. J. Virol. 74 (2000) 2293–2304.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Philip Hartjen.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hartjen, P., Höchst, B., Heim, D. et al. The NTPase/helicase domain of hepatitis C virus nonstructural protein 3 inhibits protein kinase C independently of its NTPase activity. Cell Mol Biol Lett 18, 447–458 (2013). https://doi.org/10.2478/s11658-013-0099-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-013-0099-7

Key words

  • Hepatitis C virus (HCV)
  • NS3 protein
  • Protein kinase C (PKC)
  • PKC isotypes
  • Protein kinase inhibitors
  • Pseudosubstrate inhibition
  • Hepatocellular carcinoma (HCC)