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Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model
Published on: April 16, 2014
Development and application of an in vitro apoptin kinase assay
Henriëtte L Lanz1, Bogdan I Florea, Mathieu H M Noteborn
1Department of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, 2333 CC Leiden, The Netherlands.
Abstract:
Apoptin, a protein derived from chicken anemia virus (CAV), induces apoptosis selectively in human tumor cells as compared with normal cells. This activity depends on phosphorylation and relocation of apoptin to the nucleus of cancer cells. Here, we describe an in vitro kinase assay that allows the biochemical characterization of apoptin kinase activity in tumor cells. The kinase phosphorylates apoptin in a strictly ATP-dependent fashion and in a broad salt range. The kinase activity is present constitutively in both cytoplasm and nucleus of various human tumor cells. Q-column chromatography showed that both cytoplasmic and nuclear fractions have identical fractionation characteristics, suggesting that the same kinase is present in both cellular compartments. Kinase activity derived from positive Q-column fractions bound to amylose-maltose-binding protein (MBP)-apoptin and could be eluted with ATP only in the presence of the cofactor Mg(2+). Apparently, unphosphorylated apoptin interacts with the kinase and is released only after phosphorylation has occurred, proving that our assay recognizes the genuine apoptin kinase. This is further corroborated by the finding that apoptin is phosphorylated in vitro at positions Thr108 and Thr107, in concert with earlier in vivo observations. Our assay excludes cyclin-dependent kinase 2 (CDK2) and protein kinase C beta (PKC-β), previously nominated by two separate studies as being the genuine apoptin kinase.
Insights
A novel in vitro kinase assay characterizes the tumor-selective protein apoptin
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Apoptin, derived from chicken anemia virus (CAV), selectively induces apoptosis in human tumor cells.
- Apoptin's tumoricidal activity is contingent upon its phosphorylation and nuclear translocation within cancer cells.
Purpose of the Study:
- To develop and characterize an in vitro kinase assay for identifying the enzyme responsible for apoptin phosphorylation.
- To biochemically analyze the kinase activity associated with apoptin phosphorylation in tumor cells.
Main Methods:
- Development of an ATP-dependent in vitro kinase assay.
- Utilizing Q-column chromatography to fractionate and characterize kinase activity from cytoplasmic and nuclear tumor cell extracts.
- Employing amylose-maltose-binding protein (MBP)-apoptin for kinase binding and elution studies.
Main Results:
- The assay confirmed constitutive kinase activity in both cytoplasmic and nuclear fractions of tumor cells, suggesting a single kinase entity.
- Kinase activity was ATP-dependent and functional across a broad salt range.
- The assay identified specific phosphorylation sites (Thr108 and Thr107) on apoptin, consistent with in vivo data.
- The developed assay excluded cyclin-dependent kinase 2 (CDK2) and protein kinase C beta (PKC-β) as the responsible kinase.
Conclusions:
- A robust in vitro kinase assay was established for the biochemical characterization of apoptin phosphorylation.
- The findings suggest the presence of a conserved apoptin kinase in both cytoplasmic and nuclear compartments of tumor cells.
- This assay provides a tool to exclude previously proposed kinases and advance the understanding of apoptin's mechanism of action in cancer therapy.
