Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

Jiang, Jie; Cole, Daryl; Westwood, Nigel; Macpherson, Lee; Farzaneh, Farzin; Mufti, Ghulam J.; Tavassoli, Mahvash; Gäken, Joop

doi:10.1158/0008-5472.can-10-1204

Cited by 28 publications

(33 citation statements)

References 46 publications

(59 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We first determined if AZD7762 was able to affect the localization of apoptin in the context of viral replication. MSB-1 cells were infected with wild-type CAV, and the localization of apoptin was examined by immunofluorescence using an anti-apoptin antibody (29). Figure 9A shows that apoptin localized to the nuclei of CAV-infected cells but that addition of AZD7762 resulted in cytoplasmic retention of the protein.…”

Section: Resultsmentioning

confidence: 99%

“…SDS-PAGE and Western blotting were performed with standard protocols using the following antibodies at the indicated dilutions: 1:1,000 mouse monoclonal anti-Flag M2 (Sigma), 1:1,000 rabbit monoclonal anti-HA (BioLegend), 1:2,000 mouse monoclonal anti-GFP (Clontech), 1:2,000 monoclonal rat anti-pSer10 histone H3 (Sigma), 1:500 monoclonal anti-pSer1981 ATM (Santa Cruz), 1:1,000 monoclonal rabbit anti-ATM (Cell Signaling), 1:1,000 monoclonal rabbit anti-Chk1 (Cell Signaling), 1:50 rabbit polyclonal anti-APC1 (described in reference 17), 1:1,000 mouse monoclonal anti-Cdc20 (Santa Cruz), 1:500 mouse monoclonal anti-Cdh1 (NeoMarkers), 1:1,000 mouse monoclonal anti-53BP1 (BD), 1:500 polyclonal rabbit anti-pSer25 53BP1 (Bethyl), 1:1,000 rabbit monoclonal anti-pThr68 Chk2 (Cell Signaling), 1:500 mouse monoclonal anti-Chk2 (Santa Cruz), 1:1,000 rabbit polyclonal anti-APC2 (BioLegend), 1:500 mouse monoclonal anti-Cdc27 (BD), 1:1,000 mouse polyclonal anti-APC7 (a gift of A. S. Turnell), 1:1,000 rabbit polyclonal anti-APC8 (Santa Cruz), 1:1,000 rabbit polyclonal anti-APC5 (a gift of A. S. Turnell), and 1:1,000 rabbit polyclonal antibodies against apoptin and phosphoapoptin (T108), which have been previously described (29), and 1:1,000 rabbit polyclonal anti-actin (Sigma). The secondary antibodies used were 1:5,000 goat polyclonal anti-mouse and mouse anti-rabbit horseradish peroxidase (HRP) (Jackson ImmunoResearch, Inc.).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

Kucharski

Sharon

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

Chicken anemia virus (CAV) is a single-stranded circular DNA virus that carries 3 genes, the most studied of which is the gene encoding VP3, also known as apoptin. This protein has been demonstrated to specifically kill transformed cells while leaving normal cells unharmed in a manner that is independent of p53 status. Although the mechanistic basis for this differential activity is unclear, it is evident that the subcellular localization of the protein is important for the difference. In normal cells, apoptin exists in filamentous networks in the cytoplasm, whereas in transformed cells, apoptin is present in the nucleus and appears as distinct foci. We have previously demonstrated that DNA damage signaling through the ataxia telangiectasia mutated (ATM) pathway induces the translocation of apoptin from the cytoplasm to the nucleus, where it induces apoptosis. We found that apoptin contains four checkpoint kinase consensus sites and that mutation of either threonine 56 or 61 to alanine restricts apoptin to the cytoplasm. Furthermore, treatment of tumor cells expressing apoptin with inhibitors of checkpoint kinase 1 (Chk1) and Chk2 causes apoptin to localize to the cytoplasm. Importantly, silencing of Chk2 rescues cancer cells from the cytotoxic effects of apoptin. Finally, treatment of virus-producing cells with Chk inhibitor protects them from virus-mediated toxicity and reduces the titer of progeny virus. Taken together, our results indicate that apoptin is a sensor of DNA damage signaling through the ATM-Chk2 pathway, which induces it to migrate to the nucleus during viral replication. IMPORTANCEThe chicken anemia virus (CAV) protein apoptin is known to induce tumor cell-specific death when expressed. Therefore, understanding its regulation and mechanism of action could provide new insights into tumor cell biology. We have determined that checkpoint kinase 1 and 2 signaling is important for apoptin regulation and is a likely feature of both tumor cells and host cells producing virus progeny. Inhibition of checkpoint signaling prevents apoptin toxicity in tumor cells and attenuates CAV replication, suggesting it may be a future target for antiviral therapy. Circoviruses are a diverse group of nonenveloped, icosahedral viruses containing circular, single-stranded DNA genomes (1, 2). These viruses have been shown to infect a wide range of hosts, and they are the causative agents of several serious diseases in animals. In particular, chicken anemia virus (CAV), a member of the genus Gyrovirus, is the etiological agent of chicken infectious anemia. CAV infects several bone marrow-derived cells, resulting in severe anemia and immunosuppression in young chickens and compromised immune response in older birds (3, 4). CAV can lead to considerable economic loss during intensive chicken farming, and control of the virus through vaccination is currently standard practice. Recently, a novel circovirus with partial homology to CAV isolated from a skin swab was designated human gyrovirus (HGyV) (5). The identificati...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

Kucharski

Sharon

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…PKCb inhibitor is an anilino-monoindolylmaleimide compound that acts as a potent, cell-permeable inhibitor of PKCb isozymes (25). PKCz/l inhibitor is a myristoylated pseudosubstrate peptide inhibitor (Myr-SIYRRGARRWRKL-OH), which consists of pseudosubstrate peptide sequence derived from PKCz/l and is a cell-permeable, reversible, substrate competitive inhibitor (26).…”

Section: Pkc Isoform Inhibitors and Inhibitor Treatmentmentioning

confidence: 99%

Reciprocal Regulation of Protein Kinase C Isoforms Results in Differential Cellular Responsiveness

Sudan

Srivastava

Pandey

et al. 2012

The Journal of Immunology

View full text Add to dashboard Cite

Immunological homeostasis is often maintained by counteractive functions of two different cell types or two different receptors signaling through different intermediates in the same cell. One of these signaling intermediates is protein kinase C (PKC). Ten differentially regulated PKC isoforms are integral to receptor-triggered responses in different cells. So far, eight PKC isoforms are reported to be expressed in macrophages. Whether a single receptor differentially uses PKC isoforms to regulate counteractive effector functions has never been addressed. As CD40 is the only receptor characterized to trigger counteractive functions, we examined the relative role of PKC isoforms in the CD40-induced macrophage functions. We report that in BALB/c mouse macrophages, higher doses of CD40 stimulation induce optimum phosphorylation and translocation of PKCα, βI, βII, and ε whereas lower doses of CD40 stimulation activates PKCδ, ζ, and λ. Infection of macrophages with the protozoan parasite Leishmania major impairs PKCα, βI, βII, and ε isoforms but enhances PKCδ, ζ, and λ isoforms, suggesting a reciprocity among these PKC isoforms. Indeed, PKCα, βI, βII, and ε isoforms mediate CD40-induced p38MAPK phosphorylation, IL-12 expression, and Leishmania killing; PKCδ and ζ/λ mediate ERK1/2 phosphorylation, IL-10 production, and parasite growth. Treatment of the susceptible BALB/c mice with the lentivirally expressed PKCδ- or ζ-specific short hairpin RNA significantly reduces the infection and reinstates host-protective IFN-γ–dominated T cell response, defining the differential roles for PKC isoforms in immune homeostasis and novel PKC-targeted immunotherapeutic and parasite-derived immune evasion strategies.

show abstract

“…17 However, certain tissues, including the liver, spleen, and some tumors allow passage of molecules up to 200 nm in diameter which can accommodate a typical drug delivery nanoparticle. 18 The size distribution of the nanoparticles was examined using a particle size and shape analyzer (CIS-100; Ankersmid). Figure 2 shows that the size range of the nanoparticles was 50-500 nm, their mean size was 186 nm, and the polydispersity index was 0.301.…”

Section: In Vitro Cytotoxicitymentioning

confidence: 99%

pH-sensitive degradable nanoparticles for highly efficient intracellular delivery of exogenous protein

Xu¹,

Wu²,

Chen³

et al. 2013

IJN

View full text Add to dashboard Cite

Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

Cited by 28 publications

References 46 publications

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

Reciprocal Regulation of Protein Kinase C Isoforms Results in Differential Cellular Responsiveness

pH-sensitive degradable nanoparticles for highly efficient intracellular delivery of exogenous protein

Contact Info

Product

Resources

About