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.
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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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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.
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10495_2015_1120_MOESM1_ESM.pptx
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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Bullenkamp, J., Gäken, J., Festy, F. et al. Apoptin interacts with and regulates the activity of protein kinase C beta in cancer cells. Apoptosis 20, 831–842 (2015). https://doi.org/10.1007/s10495-015-1120-6
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DOI: https://doi.org/10.1007/s10495-015-1120-6