Proteolytic cleavage of cyclin E leads to inactivation of associated kinase activity and amplification of apoptosis in hematopoietic cells.
Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G(1) to S in mammalian cells and has an established role in oncogenesis. Here we examined the role of deregulated cyclin E expression in apoptosis. The levels of p50-cyclin E initially increased, and this was followed by a decrease starting at 8 h after treatment with genotoxic stress agents, such as ionizing radiation. This pattern was mirrored by the cyclin E-Cdk2-associated kinase activity and a time-dependent expression of a novel p18-cyclin E. p18-cyclin E was induced during apoptosis triggered by multiple genotoxic stress agents in all hematopoietic tumor cell lines we have examined. The p18-cyclin E expression was prevented by Bcl-2 overexpression and by the general caspase and specific caspase 3 pharmacologic inhibitors zVAD-fluoromethyl ketone (zVAD-fmk) and N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), indicating that it was linked to apoptosis. A p18-cyclin E(276-395) (where cyclin E(276-395) is the cyclin E fragment containing residues 276 to 395) was reconstituted in vitro, with mutagenesis experiments, indicating that the caspase-dependent cleavage was at amino acid residues 272 to 275. Immunoprecipitation analyses of the ectopically expressed cyclin E(1-275), cyclin E(276-395) deletion mutants, and native p50-cyclin E demonstrated that caspase-mediated cyclin E cleavage eliminated interaction with Cdk2 and therefore inactivated the associated kinase activity. Overexpression of cyclin E(276-395), but not of several other cyclin E mutants, specifically induced phosphatidylserine exposure and caspase activation in a dose-dependent manner, which were inhibited in Bcl-2-overexpressing cells or in the presence of zVAD-fmk. Apoptosis and generation of p18-cyclin E were significantly inhibited by overexpressing the cleavage-resistant cyclin E mutant, indicating a functional role for caspase-dependent proteolysis of cyclin E for apoptosis of hematopoietic tumor cells.
Mazumder
Suparna
S
Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Gong
Bendi
B
Chen
Quan
Q
Drazba
Judith A
JA
Buchsbaum
Jeffrey C
JC
Almasan
Alexandru
A
D017209:Apoptosis / Q000528:radiation effects; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D020169:Caspases / Q000378:metabolism; D019927:Cyclin E / Q000737:chemistry / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000473:pathology* / Q000528:radiation effects; D006801:Humans; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
D019927:Cyclin E; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D020169:Caspases