title: Development and application of an in vitro apoptin kinase assay.
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.
authors.affiliation: Department of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, 2333 CC Leiden, The Netherlands.
authors.forename: Henriëtte L
authors.initials: HL
authors.lastname: Lanz
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Florea
authors.forename: Mathieu H M
authors.initials: MH
authors.lastname: Noteborn
authors.forename: Claude
authors.initials: C
authors.lastname: Backendorf
mesh_terms: D021581:Active Transport, Cell Nucleus; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D019169:Jurkat Cells; D007700:Kinetics; D013058:Mass Spectrometry; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000737:chemistry
chemical_list: D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; C091717:VP3 protein, Chicken anemia virus; D013912:Threonine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011493:Protein Kinase C; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2