Apoptosis

, Volume 20, Issue 6, pp 831–842 | Cite as

Apoptin interacts with and regulates the activity of protein kinase C beta in cancer cells

  • Jessica Bullenkamp
  • Joop Gäken
  • Frederic Festy
  • Ee Zhuan Chong
  • Tony Ng
  • Mahvash Tavassoli
Original Paper
First Online:

Abstract

Apoptin, the VP3 protein from chicken anaemia virus (CAV), induces tumour cell-specific cell death and represents a potential future anti-cancer therapeutic. In tumour but not in normal cells, Apoptin is phosphorylated and translocates to the nucleus, enabling its cytotoxic activity. Recently, the β isozyme of protein kinase C (PKCβ) was shown to phosphorylate Apoptin in multiple myeloma cell lines. However, the exact mechanism and nature of interaction between PKCβ and Apoptin remain unclear. Here we investigated the physical and functional link between PKCβ and CAV-Apoptin as well as with the recently identified Apoptin homologue derived from human Gyrovirus (HGyV). In contrast to HCT116 colorectal cancer cells the normal colon mucosa cell lines expressed low levels of PKCβI and showed reduced Apoptin activation, as evident by cytoplasmic localisation, decreased phosphorylation and lack of cytotoxic activity. Co-immunoprecipitation and proximity ligation assay studies identified binding of both CAV- and HGyV-Apoptin to PKCβI in HCT116 cells. Using Apoptin deletion constructs the N-terminal domain of Apoptin was found to be required for interacting with PKCβI. FRET-based PKC activity reporter assays by fluorescence lifetime imaging microscopy showed that expression of Apoptin in cancer cells but not in normal cells triggers a significant increase in PKC activity. Collectively, the results demonstrate a novel cancer specific interplay between Apoptin and PKCβI. Direct interaction between the two proteins leads to Apoptin-induced activation of PKC and consequently activated PKCβI mediates phosphorylation of Apoptin to promote its tumour-specific nuclear translocation and cytotoxic function.

Keywords

Chicken anaemia virus Apoptin Protein kinase C FRET Novel therapeutic agents 

Abbreviations

CAV

Chicken anaemia virus

PKC

Protein kinase C

HGyV

Human Gyrovirus

Thr-108

Threonine-108

PLA

Proximity ligation assay

CKAR

C kinase activity reporter

FRET

Fluorescence resonance energy transfer

TCSPC

Time-correlated single-photon counting

FLIM

fluorescence lifetime imaging microscopy

CFP/YFP/GFP/RFP

Cyan/yellow/green/red fluorescent protein

Cdk2

Cyclin-dependent kinase 2

NF-κB

Nuclear factor kappa B

ANOVA

Analysis of variance

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Notes

Acknowledgments

J.B. is funded by a studentship awarded by the Rosetrees Trust and student support from the Dental Institute, King’s College London. We would like to thank Professor Alexandra Newton, University of California, San Diego, for many helpful discussions and provision of constructs during the course of these studies. We are grateful to the Nikon Imaging Centre at King’s College London for providing access and help with confocal microscopes as well as the Biomedical Research Centre Flow Core Facility for assistance with flow cytometry. We would also like to thank Dr. Nina Raulf, King’s College London, for assistance with setting up and optimising the proximity ligation assay.

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10495_2015_1120_MOESM1_ESM.pptx (695 kb)
Correlation of PKCβI Levels and Apoptin Activity in HCT116 and NCM356 Cells. (a) PKCβI and PKCβII expression levels in HCT116, NCM356 and NCM460 cells were determined by Western blot analysis. (b) HCT116 and NCM356 cells were infected with Lenti-GFP (GFP) or Lenti-CAV-FLAG-AP (Apo) at an MOI of 5. Total cell lysates were collected after 2 days for Western blot analysis with the indicated antibodies; blots were cut and combined at the indicated line. (c) HCT116 and NCM356 cells were transfected with CAV-FLAG-AP, fixed after 24 hours and stained with a primary mouse anti-FLAG and secondary FITC-conjugated anti-mouse antibody. Nuclei were detected by counterstaining with DAPI. Representative fluorescence images are shown (magnification 60x, bar = 10 µm). (d) HCT116 and NCM356 cells were infected with Ad-GFP or Ad-Apoptin at an MOI of 10 and collected at 40 hours post-infection for cell death detection by Annexin V/PI staining and FACS analysis. Bars represent the percentage of Annexin V positive cells; error bars indicate SEM (n = 4). Significance was determined by two-way ANOVA (*** P < 0.001). (PPTX 695 kb)

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Jessica Bullenkamp
    • 1
  • Joop Gäken
    • 2
  • Frederic Festy
    • 3
  • Ee Zhuan Chong
    • 3
  • Tony Ng
    • 4
  • Mahvash Tavassoli
    • 1
  1. 1.Department of Molecular OncologyKing’s College LondonLondonUK
  2. 2.Department of Haemato-Oncology, Rayne InstituteKing’s College LondonLondonUK
  3. 3.Biomaterials, Biomimetics & Biophotonics Department, Dental InstituteKing’s College LondonLondonUK
  4. 4.Cancer Cell Biology & Imaging, New Hunt’s HouseKing’s College LondonLondonUK

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