Apoptin interacts with and regulates the activity of protein kinase C beta in cancer cells
Jessica Bullenkamp1, Joop Gäken, Frederic Festy
1Department of Molecular Oncology, King's College London, Guy's Campus, Hodgkin Building, London, SE1 1UL, UK.
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.
Insights
Apoptin protein triggers tumor cell death by interacting with Protein Kinase C beta (PKCβ). This interaction activates PKCβ, which then phosphorylates Apoptin, driving its nuclear translocation and anti-cancer activity specifically in tumor cells.
Area of Science:
- Molecular Biology
- Cancer Research
- Virology
Background:
- Apoptin, a protein from chicken anemia virus (CAV), exhibits tumor-specific cell death induction, making it a potential anti-cancer therapeutic.
- Apoptin's cytotoxic activity relies on phosphorylation and nuclear translocation, which occurs in tumor cells but not normal cells.
- Protein Kinase C beta (PKCβ) has been identified as a kinase that phosphorylates Apoptin in multiple myeloma cells, but the interaction mechanism is unclear.
Purpose of the Study:
- To investigate the physical and functional link between PKCβ and Apoptin from CAV and human Gyrovirus (HGyV).
- To elucidate the cancer-specific mechanism of Apoptin activation and its interaction with PKCβI.
Main Methods:
- Co-immunoprecipitation and proximity ligation assays to detect protein-protein interactions.
- Apoptin deletion constructs to identify interaction domains.
- Förster Resonance Energy Transfer (FRET)-based PKC activity reporter assays using fluorescence lifetime imaging microscopy (FLIM).
Main Results:
- Both CAV-Apoptin and HGyV-Apoptin bind to PKCβI in HCT116 colorectal cancer cells, with the N-terminal domain of Apoptin being crucial for this interaction.
- Normal colon mucosa cells express lower levels of PKCβI, showing reduced Apoptin phosphorylation, nuclear translocation, and cytotoxic activity compared to cancer cells.
- Apoptin expression significantly increases PKC activity in cancer cells but not in normal cells, indicating a cancer-specific activation pathway.
Conclusions:
- A novel, cancer-specific interplay between Apoptin and PKCβI has been demonstrated.
- Direct interaction between Apoptin and PKCβI leads to Apoptin-induced PKC activation.
- Activated PKCβI mediates Apoptin phosphorylation, promoting its nuclear translocation and subsequent tumor-specific cytotoxic function.
More Related Videos
15:43Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
Published on: January 7, 2013
09:18Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death
Published on: December 27, 2016
Related Concept Videos
01:31The Intrinsic Apoptotic Pathway
01:22PI3K/mTOR/AKT Signaling Pathway
03:03mTOR Signaling and Cancer Progression
The mTOR pathway or the...
01:17The Extrinsic Apoptotic Pathway
02:34Inhibition of Cdk Activity
02:34Inhibition of CDK Activity